CN112742360B

CN112742360B - Polyethyleneimine-silicon dioxide microsphere with surface modified with titanium phosphate/zirconium group and preparation and application thereof

Info

Publication number: CN112742360B
Application number: CN202011631596.7A
Authority: CN
Inventors: 薛芸; 张志永; 陆嘉伟; 胡哲; 舒烈波
Original assignee: Shanghai Deer Biotechnology Co ltd
Current assignee: Shanghai Deer Biotechnology Co ltd
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2021-08-03
Anticipated expiration: 2040-12-31
Also published as: CN112742360A

Abstract

The invention discloses a preparation method of polyethyleneimine-silicon dioxide microspheres with titanium phosphate/zirconium phosphate groups modified on the surfaces, which comprises the following steps: bonding polyethyleneimine molecules with high-density amino on the surface of the silica microsphere; converting a large amount of amino active hydrogen in a polyethyleneimine structure into phosphorous acid groups through a Mannich reaction; and (3) adsorbing titanium ions or zirconium ions by utilizing the chelation between phosphorous acid groups and metal ions, and finally preparing the polyethyleneimine-silicon dioxide microsphere with the surface modified with titanium phosphate/zirconium groups. The polyethyleneimine-silicon dioxide microsphere with the surface modified by the titanium phosphate/zirconium group prepared by the preparation method provided by the invention can realize specific adsorption and high-efficiency enrichment of phospholipid compounds in a complex sample matrix, can be applied to specific high-efficiency solid-phase extraction of profile phospholipid in serum, and is expected to be applied to screening of phospholipid disease markers in disease metabonomics.

Description

Polyethyleneimine-silicon dioxide microsphere with surface modified with titanium phosphate/zirconium group and preparation and application thereof

Technical Field

The invention belongs to the technical field of functionalized inorganic materials and analysis, and relates to a polyethyleneimine-silicon dioxide microsphere with a surface modified with a titanium phosphate/zirconium group, and preparation and application thereof, in particular to application thereof in enrichment, purification and separation of phospholipid molecules.

Background

Phospholipids (PLs) are a class of phosphate-containing lipids in the structure, mostly phosphoric acid (H)₃PO₄) Esters, also a few, are phosphorous acid (H)₃PO₃) Esters, the current organismsThere are two main classes of phospholipids found in (a): glycerophospholipids consisting of glycerol and sphingomyelins. The main chain of the glycerophospholipid is glycerol-3-phospholipid, namely two hydroxyl groups at sn-1 and sn-2 positions in glycerol molecules are esterified by fatty acid chains, and a phosphate group at the sn-3 position can be subjected to esterification reaction with other types of small molecular alcohols, so that other various types of glycerophosphate structures can be obtained. Sphingomyelin or dihydrosphingosine is an amino diol with a long aliphatic chain. Sphingosine or sphinganine has a hydrophobic tail composed of long-chain aliphatic hydrocarbon groups and a polar head composed of two hydroxyl groups and an amino group. Sphingomyelin contains phosphoric acid, the terminal hydrocarbyl substituent of which is phosphocholine acylethanolamine. Sphingomyelin, the most abundant in the human body, is sphingomyelin, which is composed of sphingosine, fatty acids, and phosphorylcholine.

It has been found that phospholipids have various biological functions in vivo: (i) as an amphoteric compound, phospholipid molecules are the major compounds that make up the cell membrane, and at the same time, function to maintain cell integrity. (ii) The fatty acid chains of sn-1 and sn-2 of phospholipid molecules can provide a variety of unsaturated fatty acids for the biosynthesis of eicosanoids (eicosanoids) required for various inflammatory processes in the body. (iii) Lecithin is an important source of choline in the body and plays an important role in homocysteine metabolism and vascular disease. (iv) The human brain can directly take in lecithin and choline from blood and quickly convert the lecithin and choline into acetylcholine, and is beneficial to improving learning ability and memory. (v) The phospholipid, especially lecithin, has antioxidant effect, can remove peroxide in blood, is beneficial to reducing the retention time of fat on the inner wall of a blood vessel, eliminates atherosclerotic plaque, and has obvious effects of preventing and treating hyperlipidemia and hypercholesterolemia. (vi) Cardiolipin is abundant in mitochondrial membranes and is closely related to mitochondrial-related physiological functions and diseases; it has been found that the biosynthesis and regeneration of lecithin in mammalian heart is closely related to Barth Syndrome. (vii) The cell membrane phospholipid has asymmetric distribution, and the phosphatidylserine on the inner membrane can turn out at the early stage of apoptosis. Under normal physiological and some pathological conditions, normal blood cells can be subjected to apoptosis or activated, and mechanisms such as interleukin-1 beta convertase activity down-regulation and cell membrane trans-membrane potential reduction enable PS to be turned from the side inside the cell membrane to the side outside the cell membrane, so that the cell membrane surface of the blood cells has PS expression. (viii) The inner membrane of the cell membrane contains a large amount of negatively charged phospholipids, mainly phosphatidylserine, which have been shown to play an important role in the coagulation process as well. In vitro experiments show that when PS is blocked, the coagulation process is obviously inhibited; when PS is added into the blood coagulation system, the blood coagulation process is greatly enhanced regardless of the activation of endogenous and exogenous coagulation factors X and prothrombin. (ix) The sphingomyelin and lecithin coexist outside the cell membrane, and researches show that the sphingomyelin and the lecithin participate in the processes of cell growth, differentiation and apoptosis. (x) Phosphatidylinositol and its hydrolysis products on the cell membrane help to convert extracellular signals into intracellular signals, which participate in the signal transduction process. In addition to the above physiological activities, phospholipids have been confirmed to be involved in lung function and the like. Therefore, the content and structural change of the phospholipid in blood and tissues have very important research values in pathological research, disease prevention and treatment and disease marker screening. In recent years, research on serum disease metabonomics discovers that phospholipid compounds with abnormal metabolism can be used as potential markers for monitoring various diseases, and simple and effective technical bases are provided for early diagnosis, prognosis treatment and the like.

At present, the extraction method for enriching phospholipid in biological samples is mainly divided into two main categories: liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Samples treated by the LLE method are usually clean and no or few external impurities are introduced, but the treatment processes of the methods are usually complex and the pH of the enriched solution needs to be controlled to obtain a good extraction effect; in addition, the LLE extraction method requires a large amount of samples, and is prone to cause large losses. The SPE method needs a small amount of samples, so that the utilization rate of the samples is effectively improved; since SPE methods often perform specific extraction for a certain class or classes of compounds, the purity of the obtained product is high. Some current methods for selective enrichment of phosphopeptides, such as metal ion affinity chromatography, have low specificity and tend to enrich for some non-phosphopeptides while enriching phosphopeptides, and thus such methods also have low specificity for phospholipid enrichment. And through the immobilized metal ion affinity chromatography with the phosphate group as a matching group, phosphopeptide can be adsorbed with high specificity, the nonspecific adsorption of non-phosphorylated peptide segments is effectively reduced, and the phosphopeptide can obtain higher specificity when being used for phospholipid enrichment.

Polyethyleneimine (PEI) is a water-soluble cationic polymer with high molecular weight, and is classified into linear and branched polymers, wherein branched polyethyleneimine is rich in primary amine, secondary amine and tertiary amine groups, has high reactivity, and is easy to chemically modify, such as being capable of easily reacting with epoxy groups, aldehyde groups, isocyanate compounds and acidic gases. The specific adsorption of phospholipid compounds is realized by Lewis acid-base action between metal ions such as titanium, zirconium and the like and phospholipids, and the special property enables the metal ions such as titanium, zirconium and the like to be widely applied to enrichment and separation of the phospholipids. The preparation method has the advantages of clear principle, simple operation, high reaction efficiency and easy obtainment of raw materials, and provides a feasible and effective preparation method for the material modified by phosphate groups or metal ions. Meanwhile, because the polyethyleneimine molecules are rich in amino active hydrogen, the content of titanium/zirconium phosphate on the surface of the material prepared by the method is high, and the extraction and enrichment efficiency of phosphorylated peptide or phospholipid is expected to be greatly improved.

Therefore, polyethyleneimine is modified on the surface of a silicon dioxide substrate with a large specific surface area, and a novel technical idea is provided for efficient selective enrichment, purification and separation of phospholipid compounds by utilizing high-density reactive amino groups on the surface of polyethyleneimine to combine with phosphate groups and utilizing the phosphate groups to chelate titanium/zirconium ions.

Disclosure of Invention

The invention aims to provide a polyethyleneimine-silica microsphere (a silica microsphere material with a surface modified by high-density titanium phosphate or zirconium phosphate) with a surface modified by titanium phosphate/zirconium groups, which is convenient to operate, has high specificity and can be used for efficiently separating, enriching and purifying phospholipid compounds, and a preparation method and application of the polyethyleneimine-silica microsphere with the surface modified by titanium phosphate/zirconium groups.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention provides a polyethyleneimine-silica microsphere with a surface modified with titanium phosphate/zirconium groups, which is prepared by taking a full-porous silica microsphere as a substrate, bonding polyethyleneimine molecules on the surface, performing phosphoric acid functionalization, and adsorbing titanium ions or zirconium ions on the surface of the polyethyleneimine-silica microsphere with phosphate groups enriched on the surface by utilizing strong chelation between the phosphate groups and the titanium ions (or zirconium ions).

Wherein, the silica microspheres are silica microspheres with different particle sizes and different pore diameters.

The particle size range of the silicon dioxide microspheres is 10-100 mu m; preferably 20 or 40 μm.

The pore diameter range of the silicon dioxide microspheres is 0-50 nm; preferably 12 nm.

The shape of the silica microspheres is spherical.

Further, the silica microspheres are silica microspheres with surfaces functionalized by epoxy propyl groups; or, silica microspheres containing other reactive groups.

Wherein the polyethyleneimine is branched polyethyleneimine with different molecular weights and degrees of polymerization.

The molecular weight of the polyethyleneimine is 10kDa-100 kDa; preferably, it is 10kDa or 70 kDa.

Wherein the titanium phosphate/zirconium phosphate group is formed by strong chelation between phosphate group and titanium ion (or zirconium ion) after the polyethyleneimine-silica microspheres are functionalized by phosphorous acid; or, other reactive functional groups derivatized titanium/zirconium phosphate groups.

The invention also provides a preparation method of the polyethyleneimine-silica microsphere with the surface modified with the titanium phosphate/zirconium phosphate group, which comprises the steps of firstly modifying the surface of the silica microsphere with epoxypropyl or other active functional groups, then bonding branched polyethyleneimine molecules with different molecular weights by utilizing the ring-opening reaction or other reaction mechanisms of the epoxypropyl so as to enable the surface of the silica microsphere to be rich in active functional groups such as primary amine, secondary amine and tertiary amine, then modifying the surface of the silica microsphere with phosphate groups by utilizing the high reaction activity of amino groups and chelating titanium/zirconium ions by utilizing the phosphate groups, thereby finally preparing the polyethyleneimine-silica microsphere with the surface modified with the titanium phosphate/zirconium phosphate group.

Specifically, the method comprises the following steps:

(1) modifying the surface of the silica microsphere with polyethyleneimine: performing modification of epoxypropyl or other active functional groups on the activated silica microspheres, then dispersing the silica microspheres with the surfaces modified with the epoxypropyl or other active functional groups in a polyethyleneimine solution, and stirring for reaction to obtain polyethyleneimine-modified silica microspheres;

(2) and (3) phosphoric acid functional modification of the polyethyleneimine modified silica microspheres: adding concentrated hydrochloric acid and phosphorous acid solution into the polyethyleneimine modified silica microspheres obtained in the step (1), stirring for reaction, adding formaldehyde solution, and continuing reflux reaction to obtain phosphoric acid functionalized polyethyleneimine modified silica microspheres;

(3) modifying titanium/zirconium ions on a silica microsphere modified by phosphoric acid functionalized polyethyleneimine: ultrasonically dispersing the phosphoric acid functionalized polyethyleneimine modified silica microspheres obtained in the step (2) in an aqueous solution containing titanium/zirconium ions, and stirring for reaction to obtain the polyethyleneimine-silica microspheres with the surface modified with titanium/zirconium phosphate groups.

In the step (1), the activation step of the silica microspheres is as follows: ultrasonically dispersing the silicon dioxide microspheres in a hydrochloric acid solution, and then carrying out condensation reflux reaction; and after reaction, carrying out suction filtration, washing and drying to obtain the activated silicon dioxide microspheres.

The silica microspheres are silica microspheres with different particle sizes, different pore diameters and different properties.

The shape of the silica microspheres is spherical.

Wherein the concentration of the hydrochloric acid solution is 12-36 wt%; preferably, it is 12 wt%.

Wherein the ultrasonic dispersion time is 1-5 min; preferably, it is 2 min.

Wherein the frequency of the ultrasonic dispersion is 10-100 kHz; preferably 40 kHz.

Wherein the reaction temperature is 90-110 ℃; preferably, it is 102 ℃.

Wherein the reaction time is 12-24 h; preferably 24 h.

Wherein the drying temperature is 40-80 ℃; preferably, it is 60 ℃.

In the step (1), the step of modifying the surface of the silica microsphere with active functional groups comprises: drying and dehydrating the activated silicon dioxide microspheres, and then ultrasonically dispersing in dehydrated toluene; then adding epoxypropyl or other active functional groups for reflux reaction; and after the reaction is finished, carrying out suction filtration, washing and drying to obtain the silicon dioxide microspheres with surface modified active functional groups.

Wherein the temperature for drying and dewatering is 60-120 ℃; preferably, it is 110 ℃.

Wherein the drying and water removing time is 6-12 h; preferably, it is 6 h.

Wherein the ultrasonic dispersion time is 1-5 min; preferably, it is 2 min.

Wherein the mass/volume ratio of the activated silica microspheres to the epoxypropyl groups or other active functional groups is 1 g: 1ml-1 g: 5ml of the solution; preferably, 1 g: 3 ml.

Wherein the reaction temperature is 100-120 ℃; preferably, it is 115 ℃.

Wherein the reaction time is 12-24 h; preferably 24 h.

Wherein the drying temperature is 40-80 ℃; preferably, it is from 45 to 80 ℃; further preferably, it is 45 ℃.

In the step (1), the polyethyleneimine is branched polyethyleneimine having different molecular weights and degrees of polymerization.

In the step (1), the concentration of the polyethyleneimine solution is 5-10 mg/ml; preferably, it is 8.75 mg/ml.

In the step (1), the content of the silica microspheres with the surface modified with the epoxypropyl groups or other active functional groups in the polyethyleneimine solution is 1g/10ml-1g/100 ml; preferably, it is 1g/40 ml.

In the step (1), the reaction temperature is 60-80 ℃; preferably, it is 65 ℃.

In the step (1), the reaction time is 12-24 h; preferably 12 h.

The method also comprises the step of ultrasonically dispersing the polyethyleneimine modified silicon dioxide microspheres by using ultrapure water before the step (2).

Wherein the time of ultrasonic dispersion is 10-100 kHz; preferably 40 kHz.

Wherein the frequency of ultrasonic dispersion is 1-5 min; preferably, it is 2 min.

In the step (2), the concentration of the concentrated hydrochloric acid is 12-36 wt%; preferably, it is 36 wt%.

In the step (2), the usage ratio of the polyethyleneimine modified silica microspheres to the concentrated hydrochloric acid to the phosphorous acid solution is 1 g: 10 ml: 1ml-1 g: 50 ml: 5ml of the solution; preferably, 1 g: 30 ml: 4 ml.

In the step (2), the concentration of the phosphorous acid solution is 20-80 wt%; preferably, it is 50 wt%.

In the step (2), the temperature of the reaction is 100-110 ℃; preferably, it is 105 ℃.

In the step (2), the reaction time is 5-20 min; preferably, it is 10 min.

In the step (2), the amount of the added formaldehyde solution is 1-5 ml; preferably, it is 2.5 ml.

In the step (2), the temperature of the reflux reaction is 100-120 ℃; preferably, it is 105 ℃.

In the step (2), the reflux reaction time is 1-2 h; preferably, it is 1.5 h.

In the step (3), the ultrasonic dispersion time is 1-5 min; preferably, it is 2 min.

In the step (3), the frequency of the ultrasonic dispersion is 10-100 kHz; preferably 40 kHz.

In the step (3), the concentration of the aqueous solution containing titanium/zirconium ions is 50-200 mM; preferably, it is 100 mM.

In the step (3), the aqueous solution containing titanium/zirconium ions specifically refers to titanium sulfate and ZrOCl₂·8H₂O。

In the step (3), the content of the phosphoric acid functionalized polyethyleneimine modified silica microspheres in the aqueous solution containing titanium/zirconium ions is 1g/10ml-1g/100 ml; preferably, it is 1g/60 ml.

In the step (3), the reaction temperature is 30-40 ℃; preferably, it is 30 ℃.

In the step (3), the reaction time is 2-3 h; preferably, it is 2 h.

In one embodiment, the method specifically comprises the steps of:

(1) activating the silicon dioxide microspheres: ultrasonically dispersing the silicon dioxide microspheres in a hydrochloric acid solution, and then carrying out constant-temperature condensation reflux reaction in an oil bath kettle; carrying out suction filtration after reaction, washing with ultrapure water and absolute ethyl alcohol, and drying the product in a vacuum drying oven to obtain activated silicon dioxide microspheres;

(2) modifying the surface of the silicon dioxide microsphere with active functional groups: drying and dehydrating the activated silicon dioxide microspheres, and then ultrasonically dispersing in dehydrated toluene; adding epoxypropyl or other active functional groups under the condition of magnetic stirring, and carrying out reflux reaction under the condition of constant-temperature oil bath; cooling to room temperature after the reaction is finished, carrying out suction filtration, washing with toluene, acetone and anhydrous ether, and drying the product in an oven;

(3) modifying the surface of the silica microsphere with polyethyleneimine: ultrasonically dispersing silica microspheres with surfaces modified with epoxypropyl and/or other active functional groups in an ethanol solution, adding a polyethyleneimine solution, and stirring and reacting at a constant temperature of 60-80 ℃ for 12-24 hours under the protection of nitrogen after ultrasonic treatment; cooling to room temperature after the reaction is finished, and centrifuging; dispersing the centrifugal solid with ultrapure water, then carrying out suction filtration, washing with ultrapure water, ethanol and anhydrous ether, and drying the product in a drying oven to obtain the polyethyleneimine-modified silicon dioxide microspheres;

(4) and (3) phosphoric acid functional modification of the polyethyleneimine modified silica microspheres: ultrasonically dispersing the polyethyleneimine modified silicon dioxide microspheres obtained in the step (3) by using ultrapure water; adding concentrated hydrochloric acid and phosphorous acid solution, stirring and reacting for 5-20min at constant temperature of 100 ℃ and 110 ℃ under the protection of nitrogen, adding 1-5ml of formaldehyde solution, and continuing to react for 1-2h to obtain the phosphoric acid functionalized polyethyleneimine modified silica microspheres;

(5) modifying titanium/zirconium ions on a silica microsphere modified by phosphoric acid functionalized polyethyleneimine: ultrasonically dispersing the phosphoric acid functionalized polyethyleneimine modified silica microspheres obtained in the step (4) in an aqueous solution containing titanium/zirconium ions, stirring for 2-3h at a constant temperature of 30-40 ℃ under the protection of nitrogen for reaction, and washing with ultrapure water and absolute ethyl alcohol after the reaction is finished to obtain the polyethyleneimine-silica microspheres with the surface modified with titanium phosphate/zirconium groups.

The invention also provides the polyethyleneimine-silicon dioxide microsphere with the surface modified with the titanium phosphate/zirconium phosphate group, which is prepared by the method.

The invention also provides the application of the polyethyleneimine-silicon dioxide microsphere with the surface modified with the titanium phosphate/zirconium phosphate group in enrichment, purification and separation of phospholipid-rich molecules.

The invention also provides the application of the polyethyleneimine-silicon dioxide microsphere with the surface modified with the titanium phosphate/zirconium phosphate group in solid phase extraction of phospholipid standard solution.

The invention also provides the application of the polyethyleneimine-silicon dioxide microsphere with the surface modified with the titanium phosphate/zirconium phosphate group in solid phase extraction in serum.

The beneficial effects of the invention include: the synthetic method is simple and effective, and the prepared material contains high-density titanium/zirconium phosphate on the surface and can selectively adsorb phospholipid molecules. The polyethyleneimine-silicon dioxide microspheres with the surface modified with the titanium phosphate/zirconium phosphate groups are used as an enrichment separation material of phospholipid compounds, so that the method realizes effective extraction of the profile phospholipid in serum, and is expected to be applied to efficient enrichment purification of phospholipid compounds in disease metabonomics.

Drawings

FIG. 1 is a schematic representation of the preparation process of zirconium phosphate modified polyethyleneimine-silica microspheres.

FIG. 2 shows a phospholipid standard solution (passing through 20 μm SiO)₂@PEI_10,000-PO₃-Zr⁴⁺And (4) separating and detecting HPLC-ELSD after solid phase extraction of the filler. The sample peaks in the figure are: 1.16:0Lyso PC; 2.18:0Lyso PC; PE (16:0/18: 1); 4.14:0 PC; 5.15:0 PC; PC (16:0/18: 1).

FIG. 3 is an infrared characterization spectrum of the phosphate zirconium (titanium) modified polyethyleneimine-silica microsphere.

FIG. 4 shows the standard curves of five phospholipid molecules in HPLC-ELSD detection (A is the peak area, C is the concentration (. mu.g/mL) of the corresponding phospholipid).

FIG. 5 shows the recovery of solid phase extraction from five different phospholipids by four solid phase extraction columns.

FIG. 6 shows the enrichment and detection of phospholipids in serum after protein precipitation by four solid-phase extraction columns in two solvent systems. Sample 1-40 μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺A solid phase extraction column, a normal hexane-isopropanol (20:80, v/v) system; sample2-40 μm SiO₂@PEI_70,000-PO₃-Ti⁴⁺A solid phase extraction column, n-hexane-isopropanol (20:80, v/v); sample 3-40 μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺/Ti⁴⁺(1+1) a solid phase extraction column, n-hexane-isopropanol (20:80, v/v); sample 4-40 μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺&Ti⁴⁺(1:1) a solid phase extraction column, n-hexane-isopropanol (20:80, v/v); sample 5-40 μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺Solid phase extraction column, acetonitrile system; sample 6-40 μm SiO₂@PEI_70,000-PO₃-Ti⁴⁺Solid phase extraction column, acetonitrile system; sample 7-40 μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺/Ti⁴⁺(1+1) a solid phase extraction column and an acetonitrile system; sample 8-40 μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺&Ti⁴⁺(1:1) a solid phase extraction column and an acetonitrile system; sample 9-serum Sample pretreated by protein precipitation; each ID number represents the characteristic parent ion mass to charge ratio (Q1, m/z) and daughter ion mass to charge ratio (Q3, m/z) of a phospholipid compound.

FIG. 7 is a 36:1PC-O chromatogram obtained by extracting an ion pair (Q1/Q3,774.7/184.1) after UPLC-QqQ MS separation and detection before and after phospholipid solid phase extraction in serum.

FIG. 8 is a 20:4Lyso PC chromatogram obtained by extracting an ion pair (Q1/Q3,544.4/184.1) after UPLC-QqQ MS separation and detection before and after phospholipid solid phase extraction in serum.

FIG. 9 is a 40:5PE chromatogram obtained by extracting ion pairs (Q1/Q3,794.7/653.7) after UPLC-QqQ MS separation and detection before and after phospholipid solid phase extraction in serum.

FIG. 10 is 20:5Lyso PE chromatogram obtained by extracting ion pairs (Q1/Q3,500.5/359.5) after UPLC-QqQ MS separation and detection before and after phospholipid solid phase extraction in serum.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

Example 1: 20 μm SiO₂@PEI_10,000-PO₃-Zr⁴⁺Preparation of microsphere materials

Full porous 20 mu m-SiO₂Activating microspheres with hydrochloric acid: weighing 1g of full-porous 20 mu m-SiO₂Placing the microspheres in a 100mL round-bottom flask, adding 20mL of 12 wt% hydrochloric acid solution, ultrasonically dispersing for 2min, placing in an oil bath, and carrying out condensation reflux reaction at 102 ℃ for 24 h. And after the reaction is finished, carrying out suction filtration, adding a large amount of ultrapure water for washing to be neutral, adding a small amount of absolute ethyl alcohol for washing, and drying the obtained solid in a vacuum drying oven at 60 ℃ for later use.

Full porous 20 mu m-SiO₂Modifying the surface of the microsphere with epoxypropyl: weighing 1g of full-porous 20 mu m-SiO₂And (3) dewatering the microspheres in a vacuum drying oven at 110 ℃ for 6 hours, transferring the microspheres into a 100mL dry round-bottom flask, adding 40mL of dewatered toluene, and performing ultrasonic treatment for 2min to uniformly disperse the microspheres. Adding 3mLKH-560 under magnetic stirring, installing a condenser tube and a nitrogen protection device, and carrying out reflux reaction for 24h under the condition of 115 ℃ oil bath. After the reaction is finished, cooling to room temperature, carrying out suction filtration, and sequentially washing with 50mL of toluene, acetone and anhydrous ether. Collecting the product after suction filtration, and drying in a 45 ℃ oven for later use to obtain 20 mu m-SiO₂-glycodyl microspheres.

20μm-SiO₂-Glycidyl microsphere surface modified PEI_10,000: 1g of the 20 μm-SiO prepared above was weighed₂Dispersing the-glycyl microspheres in 40mL of ethanol solution, performing ultrasonic treatment for 2min to uniformly disperse the microspheres, and adding 40mL of PEI_10,000Aqueous solution (weighing PEI_10,0000.35g of liquid, 40mL of ultrapure water was added, and the mixture was stirred and dispersed). After ultrasonic treatment for 2min, placing in an oil bath pan, starting magnetic stirring, installing a condensing device and a nitrogen protection device, and reacting for 12h at 65 ℃. After the reaction was completed, the reaction mixture was cooled to room temperature, centrifuged, and the supernatant was discarded. The solid was dispersed again in ultrapure water, filtered, and washed with 30mL each of ultrapure water, ethanol and dehydrated ether in this order. Collecting the product after suction filtration, and drying in a 45 ℃ oven for later use to obtain 20 mu m-SiO₂@PEI_10,000And (3) microspheres.

20μm-SiO₂@PEI_10,000Phosphorylation of microspheres: weighing 20 μm-SiO₂@PEI_10,000Adding 1g of microspheres into a round-bottom flask, adding 30ml of ultrapure water, and performing ultrasonic dispersion for 2 min; under the condition of magnetic stirring, 30mL of concentrated hydrochloric acid (36 wt%) and 4mL of phosphorous acid solution (50 wt%) are sequentially added, a nitrogen protection device is installed, and the temperature is raisedMaintaining at 105 deg.C for 10 min; 2.5mL of formaldehyde solution was added dropwise to the round-bottom flask using a syringe and the reaction was continued at reflux for 1.5 h. After the reaction is finished, adding a large amount of degassed ultrapure water to terminate the reaction, performing suction filtration, washing with ultrapure water to be neutral, then adding a small amount of absolute ethyl alcohol to wash, and draining for later use to obtain the SiO with the particle size of 20 mu m₂@PEI_10,000-PO₃H₂And (3) microspheres.

The 20 μm SiO₂@PEI_10,000-PO₃H₂The microspheres need to be stored in an oxygen-free environment to prevent oxidation.

20μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺Preparation of the filler: weighing 20 μm SiO₂@PEI_10,000-PO₃H₂1.0g of microspheres, 60mLZrOCl was added₂Ultrasonic dispersing for 2min in 100mM water solution, placing in 30 deg.C oil bath under nitrogen protection, and reacting for 2h under magnetic stirring. After the reaction is finished, the reaction solution is filtered, and a large amount of ultrapure water is added for washing to remove excessive Zr⁴⁺Adding a small amount of absolute ethyl alcohol, washing and draining for later use to obtain 20 mu m SiO₂@PEI_10,000-PO₃-Zr⁴⁺And (4) filling.

Example 2: 20 μm SiO₂@PEI_10,000-PO₃-Zr⁴⁺Microsphere material for solid phase extraction of phospholipid standard substance solution

In order to preliminarily verify the feasibility of the phosphate (IV) valence metal ion group modified polyethyleneimine-silicon dioxide microspheres prepared by the method in phospholipid extraction, the invention firstly prepares the SiO with the thickness of 20 mu m₂@PEI_10,000-PO₃-Zr⁴⁺The filler is applied to the solid phase extraction of 6 phospholipid standards. And (3) spin-drying effluent liquid obtained after sample loading, leaching and elution, and detecting the spin-dried solid by using an HPLC-ELSD system after redissolving the solid by using methanol.

Preparing a phospholipid standard substance solution: an appropriate amount of phospholipid standard substance was dissolved in a methanol solution to prepare a phospholipid standard substance mixture solution (i) containing 0.71mM PC (16:0/18:1), 0.50mM 15:0PC, 0.18mM PE (16:0/18:1), 0.63mM 16:0Lyso PC, 0.80mM 14:0PC and 0.89mM 18:0Lyso PC.

20μm SiO₂@PEI_10,000-PO₃-Zr⁴⁺Preparing a solid phase extraction column: weighing and drying 20 mu m SiO₂@PEI_10,000-PO₃-Zr⁴⁺Filling 30mg of microspheres into a 1mL polypropylene solid phase extraction hollow column tube, and placing polyethylene sieve plates at two ends of the filler.

Solid phase extraction process of phospholipid standard substance solution (I): (1) pretreatment: 1mL of a solution of X2 ammonia-methanol (0.1M), 1mL of a solution of X2 n-hexane in isopropanol (20:80, v/v) and 0.5mL of a solution of X2 n-hexane in isopropanol in trifluoroacetic acid (20:80:0.5, v/v) were added in this order to prepare a solution of the above-mentioned 20 μ M SiO₂@PEI_10,000-PO₃-Zr⁴⁺Pretreating a solid phase extraction column; (2) loading: taking 100 mu L of the pre-prepared phospholipid standard substance mixed solution, dispersing the mixed solution into 900 mu L of n-hexane, isopropanol and trifluoroacetic acid solution (20:80:0.5, v/v), transferring the mixed solution to the upper end of a solid phase extraction column filler, keeping the vacuum degree of 30kPa, enabling a sample Loading solution to pass through the solid phase extraction column, and marking the obtained liquid as Loading; (3) leaching: adding 0.5mL of an isopropanol solution (20:80, v/v) of x 4 n-hexane, and collecting the obtained liquid as Washing; (4) and (3) elution: 0.3mL of 5 ammonia-methanol solution (0.1M) was added for Elution, and the collected liquid was labeled as Elution. (5) Redissolving: and (3) carrying out centrifugal concentration and drying on the collected three liquids of Loading, Washing and Elution, adding 100 mu L of methanol into the obtained solid, carrying out re-dissolution by vortex for 1min, carrying out high-speed centrifugation (12000rpm,5min) on the obtained sample, and taking the supernatant for subsequent detection.

HPLC-ELSD detection conditions: kromasil C8 column (5 μm, 250X 4.6 mm); sample introduction amount: 10 mu L of the solution; mobile phase A: methanol-water solution containing 10mM ammonium acetate (80:20, v/v), mobile phase B: a methanol solution containing 10mM ammonium acetate; the gradient conditions were: 0-3min, 50% B/3-18min, 50-100% B/18-22min, 100% B; the temperature of an ELSD evaporating pipe is 45 ℃; the atomizing gas is air; the gas flow rate was 2.5L/min.

As can be seen from the results in FIG. 2, no three types of phospholipids, PC, Lyso PC and PE were detected in the effluent after loading and washing, while 6 types of phospholipid standards before extraction were detected in the effluent after elution. The result shows that the polyethyleneimine-silicon dioxide microspheres modified by phosphate (IV) valence metal ion (titanium or zirconium) groups prepared by the method have specific selective adsorption capacity on phospholipid molecules and can realize rapid elution.

Example 3: 40 μm SiO₂@PEI_10,000-PO₃-Zr⁴⁺(Ti⁴⁺Or Zr⁴⁺&Ti⁴⁺) Preparation of microsphere materials

Full porous 40 mu m-SiO₂Activating microspheres with hydrochloric acid: 1g of all-porous 40 μm-SiO was weighed₂Placing the microspheres in a 100mL round-bottom flask, adding 20mL of 12 wt% hydrochloric acid solution, ultrasonically dispersing for 2min, placing in an oil bath, and carrying out condensation reflux reaction at 102 ℃ for 24 h. And after the reaction is finished, carrying out suction filtration, adding a large amount of ultrapure water for washing to be neutral, adding a small amount of absolute ethyl alcohol for washing, and drying the obtained solid in a vacuum drying oven at 60 ℃ for later use.

Full porous 40 mu m-SiO₂Modifying the surface of the microsphere with epoxypropyl: 1g of all-porous 40 μm-SiO was weighed₂And (3) dewatering the microspheres in a vacuum drying oven at 110 ℃ for 6 hours, transferring the microspheres into a 100mL dry round-bottom flask, adding 40mL of dewatered toluene, and performing ultrasonic treatment for 2min to uniformly disperse the microspheres. Adding 3mLKH-560 under magnetic stirring, installing a condenser tube and a nitrogen protection device, and carrying out reflux reaction for 24h under the condition of 115 ℃ oil bath. After the reaction is finished, cooling to room temperature, carrying out suction filtration, and sequentially washing with 50mL of toluene, acetone and anhydrous ether. Collecting the product after suction filtration, and drying the product in a 45 ℃ oven for later use to obtain 40 mu m-SiO₂-glycodyl microspheres.

40μm-SiO₂-Glycidyl microsphere surface modified PEI_70,000: 1g of the 40 μm-SiO prepared above was weighed₂Dispersing the-glycyl microspheres in 40mL of ethanol solution, performing ultrasonic treatment for 2min to uniformly disperse the microspheres, and adding 40mL of PEI_70,000Aqueous solution (weighing PEI_70,0000.7g of the solution (50 wt%) was added to 40mL of ultrapure water, and the mixture was dispersed with stirring. After ultrasonic treatment for 2min, placing in an oil bath pan, starting magnetic stirring, installing a condensing device and a nitrogen protection device, and reacting for 12h at 65 ℃. After the reaction was completed, the reaction mixture was cooled to room temperature, centrifuged, and the supernatant was discarded. Dispersing the solid again with ultrapure water, filtering, and sequentiallyThe resulting solution was washed with 30mL each of ultrapure water, ethanol and dehydrated ether. Collecting the product after suction filtration, and drying the product in a 45 ℃ oven for later use to obtain 40 mu m-SiO₂@PEI_70,000And (3) microspheres.

40μm-SiO₂@PEI_70,000Phosphorylation of microspheres: weighing 40 mu m-SiO₂@PEI_70,0001g of microspheres are added into a round-bottom flask, 30mL of ultrapure water is added, and ultrasonic dispersion is carried out for 2 min; under the condition of magnetic stirring, sequentially adding 30mL of concentrated hydrochloric acid (36 wt%) and 4.8mL of phosphorous acid solution (50 wt%), installing a nitrogen protection device, heating to 105 ℃, and keeping for 10 min; 3mL of formaldehyde solution was added dropwise to the round-bottom flask using a syringe, and the reaction was continued under reflux for 1.5 h. After the reaction is finished, adding a large amount of degassed ultrapure water to terminate the reaction, performing suction filtration, washing with ultrapure water to be neutral, then adding a small amount of absolute ethyl alcohol to wash, and draining for later use to obtain 40 mu m SiO₂@PEI_70,000-PO₃H₂And (3) microspheres.

The 40 μm SiO₂@PEI_10,000-PO₃H₂The microspheres need to be stored in an oxygen-free environment to prevent oxidation.

40μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺Preparation of the filler: weighing 40 μm SiO₂@PEI_70,000-PO₃H₂1.0g of microspheres, 100ml of LZrOCl was added₂Ultrasonic dispersing for 2min in 100mM water solution, placing in 30 deg.C oil bath under nitrogen protection, and reacting for 2h under magnetic stirring. After the reaction is finished, the reaction solution is filtered, and a large amount of ultrapure water is added for washing to remove excessive Zr⁴⁺Adding a small amount of absolute ethyl alcohol, washing and draining for later use to obtain 40 mu m SiO₂@PEI_70,000-PO₃-Zr⁴⁺And (4) filling.

40μm SiO₂@PEI_70,000-PO₃-Ti⁴⁺Preparation of the filler: weighing 40 μm SiO₂@PEI_70,000-PO₃H₂1.0g of microspheres, 100ml of Ti (SO) was added₄)₂Ultrasonic dispersing for 2min in 100mM water solution, placing in 30 deg.C oil bath under nitrogen protection, and reacting for 2h under magnetic stirring. After the reaction is finished, the reaction solution is filtered, and a large amount of the solution is addedUltra pure water washing to remove excess Ti⁴⁺Adding a small amount of absolute ethyl alcohol, washing and draining for later use to obtain 40 mu m SiO₂@PEI_70,000-PO₃-Ti⁴⁺And (4) filling.

40μm SiO2@PEI_70,000-PO₃-Zr⁴⁺&Ti⁴⁺(1:1) preparation of Filler: weighing 40 μm SiO₂@PEI_70,000-PO₃H₂1.0g of microspheres, 100mL of Ti (SO) containing₄)₂And ZrOCl₂Ultrasonic dispersing 50mM of each aqueous solution for 2min, placing the mixture in an oil bath kettle at the temperature of 30 ℃ under the protection of nitrogen, and reacting for 2h under the condition of magnetic stirring. After the reaction is finished, the reaction solution is filtered, and a large amount of ultrapure water is added for washing to remove excessive Zr⁴⁺And Ti⁴⁺Adding a small amount of absolute ethyl alcohol, washing and draining for later use to obtain 40 mu m SiO₂@PEI_70,000-PO₃-Zr⁴⁺&Ti⁴⁺(1:1) a filler.

The phosphate zirconium (titanium) modified polyethyleneimine-silica microsphere and the intermediate product in the preparation process are subjected to infrared chromatographic characterization, and the result is shown in figure 3. The results showed that the characteristic peak due to the phosphoester bond and the Si-O stretching vibration characteristic peak region (1000-^-1) Therefore, the success of the bonding of the phosphate ester bond cannot be characterized. However, at 500-^-1And 2800-^-1The infrared absorption peaks in the two regions have relatively obvious changes, and the changes of the functional groups on the surfaces of different microspheres are reflected to a certain extent.

Example 4: preparation of four solid-phase extraction columns

1)40μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺Manufacturing a solid phase extraction column: weighing and drying 40 mu m SiO₂@PEI_70,000-PO₃-Zr⁴⁺30mg of microspheres are filled into a 1mL polypropylene solid phase extraction hollow column tube, and polyethylene sieve plates are arranged at two ends of the filler.

2)40μm SiO₂@PEI_70,000-PO₃-Ti⁴⁺Manufacturing a solid phase extraction column: weighing and drying 40 mu m SiO₂@PEI_70,000-PO₃-Ti⁴⁺30mg of microspheres are filled into a 1mL polypropylene solid phase extraction hollow column tube, and polyethylene sieve plates are arranged at two ends of the filler.

3)40μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺&Ti⁴⁺(1:1) preparation of solid phase extraction column: weighing and drying 40 mu m SiO₂@PEI_70,000-PO₃-Zr⁴⁺&Ti⁴⁺(1:1) mixing 30mg of microspheres uniformly, filling the mixture into a 1mL polypropylene solid phase extraction hollow column tube, and placing polyethylene sieve plates at two ends of the filler.

4)40μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺/Ti⁴⁺(1+1) preparation of solid phase extraction column: weighing and drying 40 mu m SiO₂@PEI_10,000-PO₃-Zr⁴⁺Microspheres and 40 μm SiO₂@PEI_70,000-PO₃-Ti⁴⁺And (4) mixing the microspheres 15mg respectively, filling the mixture into a 1mL polypropylene solid phase extraction hollow column tube, and placing polyethylene sieve plates at two ends of the filler.

Example 5: 40 μm SiO₂@PEI_10,000-PO₃-Zr⁴⁺(Ti⁴⁺Or Zr⁴⁺&Ti⁴⁺) Microsphere material for solid phase extraction of phospholipid standard substance solution

Preparing a phospholipid standard substance solution: a proper amount of phospholipid standard substance is weighed and dissolved in methanol solution to prepare phospholipid standard substance mixed solution (II) containing 2.20mg/mL of 14:0 lecithin (PC), 1.62mg/mL of 16:0 Lysolecithin (LPC), 0.21mg/mL of 16:0 cephalin (PE), 0.8mg/mL of Phosphatidylglycerol (PG) (16:0/18:1) and 1.07mg/mL of 18:0 Lysocephalin (LPE).

Setting of a standard curve: diluting phospholipid standard substance solution with methanol to obtain 1-fold, 2-fold, 4-fold, 8-fold and 16-fold solutions, sampling 10 μ L of each sample, detecting with HPLC-ELSD, and making standard curve of peak area logarithm and concentration logarithm.

Solid phase extraction process of phospholipid standard solution II: (1) pretreatment: the four titanium (zirconium) phosphate groups were modified by adding 1mL of a solution of X2 ammonia-methanol (0.1M), 1mL of a solution of X2 n-hexane in isopropanol (20:80, v/v) and 0.5mL of a solution of X2 n-hexane in isopropanol in trifluoroacetic acid (20:80:0.5, v/v) in this orderOf 40 μm SiO₂@PEI_70,000Pretreating a solid phase extraction column; (2) loading: taking 100 mu L of the pre-prepared phospholipid standard substance mixed solution of different dilution times, dispersing the pre-prepared phospholipid standard substance mixed solution to 900 mu L of n-hexane, isopropanol, trifluoroacetic acid solution (20:80:0.5, v/v), transferring the solution to the upper end of a solid phase extraction column filler, keeping the vacuum degree of 30kPa, enabling a sample Loading solution to pass through the solid phase extraction column, and sequentially marking the liquid obtained by the four solid phase extraction columns as Loading-1, Loading-2, Loading-3 and Loading-4; (3) leaching: adding 0.5mL of an isopropanol solution (20:80, v/v) of X4 n-hexane, and collecting the obtained liquid marks of Washing-1, Washing-2, Washing-3 and Washing-4 respectively; (4) and (3) elution: 0.3mL of 5 ammonia-methanol solution (0.1M) was added for Elution, and the collected liquids were labeled as Elution-1, Elution-2, Elution-3 and Elution-4. (5) Redissolving: and respectively carrying out centrifugal concentration and drying on the collected loading, leaching and eluting liquids of the 4 solid-phase extraction columns, adding 100 mu L of methanol into the obtained solid, carrying out re-dissolution by vortex for 1min, carrying out high-speed centrifugation (12000rpm,5min) on the obtained sample, and then taking the supernatant for subsequent detection.

HPLC-ELSD detection conditions: kromasil C8 column (5 μm, 250X 4.6 mm); sample introduction amount: 10 mu L of the solution; mobile phase A: methanol-water solution containing 10mM ammonium acetate (80:20, v/v), mobile phase B: a methanol solution containing 10mM ammonium acetate; the gradient conditions were: 0-3min, 50% B/3-18min, 50-100% B/18-22min, 100% B; the flow rate is 0.4 ml/min; the column temperature was 55 ℃; the temperature of an ELSD evaporating pipe is 35 ℃; the atomizing gas is air; the gas flow rate was 2.5L/min.

Most phospholipid structures have no chromophoric groups or have small ultraviolet absorption wavelength and low sensitivity, so that the invention utilizes a general HPLC-ELSD separation detection system to measure a series of phospholipid standard substance mixed solutions with different concentrations, and makes log-log curves for chromatographic peak areas and phospholipid concentrations to obtain linear regression standard curves of five standard phospholipid molecules, as shown in figure 4. Therefore, the five phospholipid standard products can obtain a good standard curve, and can be applied to the determination of the recovery rate of the phospholipid after solid-phase extraction.

The extraction recovery of five different phospholipid standards by four different types of solid phase extraction columns was calculated according to the following formula, and the results are shown in FIG. 5.

Wherein Recovery is the solid phase extraction Recovery rate of different phospholipid standard substances, C_SPEAfter solid phase extraction, the concentration (mu g/mL) of different phospholipids is obtained according to the corresponding linear regression curve, V_SPEReconstituted volume (μ L), C₀Concentrations of various phospholipids (μ g/mL), V, prior to loading₀The loading volume (. mu.L).

As can be seen from the HPLC-ELSD of FIG. 5, the recovery rates for different types of phospholipids also differ for different types of solid phase extraction columns: 40 μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺The filler has high recovery rate to PC, LPC, PE and Lyso PE, but has low recovery rate to PG; and 40 μm SiO₂@PEI_70,000-PO₃-Ti⁴⁺Filler and 40 μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺Compared with the filler, the recovery rate of the PC, Lyso PC, PE and Lyso PE is reduced, but the recovery rate of the PG is improved; furthermore, 40 μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺&Ti⁴⁺(1:1) Filler and 40 μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺/Ti⁴⁺(1+1) Filler did not achieve bonding to 40 μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺And 40 μm SiO₂@PEI_70,000-PO₃-Ti⁴⁺The purpose of the two fillers was to enrich the different phospholipids, on the contrary, the recovery capacity was reduced for 5 types of phospholipids, in particular 40 μm SiO₂@PEI_70,000-PO₃-Zr⁴⁺&Ti⁴⁺(1:1) Filler, the recovery was lowest for all five of these types.

Example 6: solid phase extraction and assay in serum

Serum sample pretreatment-protein precipitation: taking a normal human serum sample out of a refrigerator at minus 80 ℃, slowly heating to melt, taking 20 mu L of the normal human serum sample, adding 180 mu L of a methanol-acetonitrile (1:1) solution containing 1 wt% of formic acid pre-cooled in a refrigerator at 4 ℃, vortexing for 2min, standing for 10min in the refrigerator at 4 ℃, then centrifuging at a high speed at a low temperature (4 ℃, 12000rpm for 10min), and taking the supernatant to obtain the serum after protein precipitation.

And (3) extracting phospholipid from serum after protein precipitation: 1) pretreatment: the four titanium (zirconium) phosphate groups were modified by sequentially adding 1mL of a solution of X2 ammonia-methanol (0.1M), 1mL of a solution of X2 n-hexane in isopropanol (20:80, v/v) (or 1mL of X2 acetonitrile) and 0.5mL of a solution of X2 n-hexane in isopropanol in trifluoroacetic acid (20:80:0.5, v/v) (or 0.5mL of X2 acetonitrile containing 0.5% trifluoroacetic acid) to the mixture to form a 40 μ M SiO solution₂@PEI_70,000Pretreating a solid phase extraction column; 2) loading: dispersing 150 mu L of the serum after the protein precipitation into 850 mu L of n-hexane, isopropanol, trifluoroacetic acid solution (20:80:0.5, v/v) (or 850 mu L of acetonitrile containing 0.5 percent of trifluoroacetic acid), transferring the serum to the upper end of a solid phase extraction column filler, keeping the vacuum degree of 30kPa, enabling a Loading solution to pass through the solid phase extraction column, and sequentially marking the liquids obtained by the four solid phase extraction columns as Loading-1, Loading-2, Loading-3 and Loading-4; 3) washing: adding 0.5mL of an isopropanol solution (20:80, v/v) of X4 n-hexane (or 0.5mL of acetonitrile of X4), and collecting the obtained liquid marks of Washing-1, Washing-2, Washing-3 and Washing-4 respectively; 4) and (3) elution: eluting with 0.3mL of 5 ammonia-methanol solution (0.1M), and collecting the collected liquids as Elution-1, Elution-2, Elution-3 and Elution-4; 5) redissolving: and respectively carrying out centrifugal concentration and drying on the collected loading, leaching and eluting liquids of the 4 solid-phase extraction columns, adding 150 mu L of methanol into the obtained solid, carrying out re-dissolution by vortex for 1min, carrying out high-speed centrifugation (12000rpm,5min) on the obtained sample, and then taking the supernatant to carry out subsequent ultra-performance liquid chromatography-mass spectrometry detection.

UPLC conditions: waters ACQUITY UPLC BEH ShieldRP18 column (1.7 μm, 2.1X 100 mm); mobile phase A: methanol-water solution containing 20mM (5:95, v/v), mobile phase B: methanol, gradient conditions see table 1; flow rate: 0.4 mL/min; column temperature: at 55 ℃.

TABLE 1 UPLC gradient elution conditions

Mass spectrum conditions: curtain gas is 15.0 psi; atomizing gas at 15.0 psi; the auxiliary gas was 10.0 psi; the ion source voltage was 4500V; the ion source temperature was 500 ℃.

Serum subjected to protein precipitation is subjected to phospholipid extraction by four different types of titanium (zirconium) phosphate solid-phase extraction columns under two solution systems (a normal hexane-isopropanol system and an acetonitrile system), UPLC-QqQ MS detection is performed after the redissolution of eluent, and hundreds of phospholipids in the serum are detected by using a Multiple Reaction Monitoring (MRM) technology, which is shown in figure 6. The results show that the four types of solid phase extraction columns can realize selective extraction of phospholipid in serum under the condition of two solvent systems. FIGS. 7, 8, 9 and 10 are chromatograms of 36:1PC-O, 20:4Lyso PC, 40:5PE and 20:5Lyso PE after extraction of ion pairs (Q1/Q3,774.7/184.1), (Q1/Q3,544.4/184.1), (Q1/Q3,794.7/653.7) and (Q1/Q3,500.5/359.5), respectively.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, which is set forth in the following claims.

Claims

1. The polyethyleneimine-silica microsphere with the surface modified with the titanium phosphate/zirconium phosphate groups is characterized in that the microsphere takes a full-porous silica microsphere as a matrix, and polyethyleneimine molecules are bonded on the surface of the full-porous silica microsphere; and converting amino active hydrogen in the polyethyleneimine structure into a phosphorous acid group, and chelating tetravalent metal ions in the phosphorous acid group.

2. The microsphere of claim 1, wherein the matrix silica microsphere is selected from silica microspheres having a particle size of 10-100 μm and a pore size of 0-50 nm; and/or the polyethyleneimine molecule is branched polyethyleneimine with the molecular weight of 10-100 kDa; and/or the tetravalent metal ions are titanium ions, zirconium ions or a mixture of the titanium ions and the zirconium ions.

3. A preparation method of polyethyleneimine-silica microspheres with titanium phosphate/zirconium phosphate groups modified on the surfaces is characterized by comprising the following steps:

4. The method according to claim 3, wherein in the step (1), the step of activating the silica microspheres comprises: ultrasonically dispersing the silicon dioxide microspheres in a hydrochloric acid solution, and then carrying out condensation reflux reaction; and after reaction, carrying out suction filtration, washing and drying to obtain the activated silicon dioxide microspheres.

5. The preparation method according to claim 4, wherein the silica microspheres have a particle size of 10-100 μm and a pore size of 0-50 nm; the concentration of the hydrochloric acid solution is 12-36 wt%; the ultrasonic dispersion time is 1-5 min; the frequency of the ultrasonic dispersion is 10k-100k Hz; the reaction temperature is 90-110 ℃; the reaction time is 12-24 h; the drying temperature is 40-80 ℃.

6. The preparation method according to claim 3, wherein in the step (1), the step of modifying the surface of the silica microspheres with active functional groups comprises: drying and dehydrating the activated silicon dioxide microspheres, and then ultrasonically dispersing in dehydrated toluene; then adding epoxypropyl or other active functional groups for reflux reaction; and after the reaction is finished, carrying out suction filtration, washing and drying to obtain the silicon dioxide microspheres with surface modified active functional groups.

7. The method for preparing the compound of claim 6, wherein the temperature for drying and removing water is 60-120 ℃; the drying and dewatering time is 6-12 h; the ultrasonic dispersion time is 1-5 min; the frequency of the ultrasonic dispersion is 10-100k Hz; the mass/volume ratio of the activated silicon dioxide microspheres to the epoxypropyl groups or other active functional groups is 1 g: 1ml-1 g: 5ml of the solution; the temperature of the reaction is 100-120 ℃; the reaction time is 12-24 h; the drying temperature is 40-80 ℃.

8. The production method according to claim 3, wherein in the step (1), the concentration of the polyethyleneimine solution is 5 to 10 mg/ml; the content of the silica microspheres with the surface modified with the epoxypropyl groups or other active functional groups in the polyethyleneimine solution is 1g/10ml-1g/100 ml; the reaction temperature is 60-80 ℃; the reaction time is 12-24 h.

9. The method according to claim 3, wherein in the step (2), the concentration of the concentrated hydrochloric acid is 12 to 36 wt%; the concentration of the phosphorous acid solution is 20-80 wt%; the dosage ratio of the polyethyleneimine modified silicon dioxide microspheres to the concentrated hydrochloric acid to the phosphorous acid solution is 1 g: 10 ml: 1ml-1 g: 50 ml: 5ml of the solution; the reaction temperature is 100-110 ℃; the reaction time is 5-20 min; the amount of the added formaldehyde solution is 1-5 ml; the temperature of the reflux reaction is 100-120 ℃; the time of the reflux reaction is 1-2 h.

10. The production method according to claim 3, wherein in the step (3), the time for the ultrasonic dispersion is 1 to 5 min; the frequency of the ultrasonic dispersion is 10-100k Hz; the concentration of the aqueous solution containing titanium/zirconium ions is 50-200 mM; the content of the phosphoric acid functionalized polyethyleneimine modified silica microspheres in the aqueous solution containing titanium/zirconium ions is 1g/10ml-1g/100 ml; the reaction temperature is 30-40 ℃; the reaction time is 2-3 h.

11. Polyethyleneimine-silica microspheres with surface-modified titanium/zirconium phosphate groups, prepared according to the method of any one of claims 3 to 10.

12. Use of the surface modified titanium phosphate/zirconium phosphate group polyethyleneimine-silica microspheres according to claim 1 or 11 for enrichment, purification and isolation of phospholipid-rich molecules.

13. Use of the surface-modified titanium/zirconium phosphate group polyethyleneimine-silica microspheres according to claim 1 or 11 in solid phase extraction of phospholipid standard solutions.

14. Use of the surface-modified titanium/zirconium phosphate group polyethyleneimine-silica microspheres according to claim 1 or 11 for solid phase extraction in serum.