CN114073941A

CN114073941A - Organic-inorganic hybrid material and preparation method and application thereof

Info

Publication number: CN114073941A
Application number: CN202110961475.7A
Authority: CN
Inventors: 徐晨; 张凯; 杨振; 徐增睿; 王明; 张雪丽; 陈武炼
Original assignee: Shanghai Anpu Experimental Technology Co ltd
Current assignee: Shanghai Anpu Experimental Technology Co ltd
Priority date: 2021-08-20
Filing date: 2021-08-20
Publication date: 2022-02-22
Anticipated expiration: 2041-08-20
Also published as: CN114073941B

Abstract

The invention discloses an organic-inorganic hybrid material and a preparation method and application thereof. The invention obtains a new organic-inorganic hybrid material by copolymerization, condensation and modification of organic-inorganic monomers, uses the new material as a solid phase extraction filler, utilizes the interaction of polar and non-polar parts of a phospholipid molecular structure to achieve the effect of removing more than 99.99 percent of phospholipid, has simple operation in the post-treatment process, can directly pass through a column after sample treatment, has low solvent consumption, is environment-friendly and the like.

Description

Organic-inorganic hybrid material and preparation method and application thereof

Technical Field

The invention relates to a high molecular material, in particular to an organic-inorganic hybrid material and a preparation method and application thereof.

Background

Phospholipid refers to lipid compound containing phosphate, and is an amphoteric molecule, and has a structure including a polar hydrophilic head end containing phosphate group on one side and a non-polar hydrophobic tail end containing two long fatty acid chains on the other side. Phospholipids are various in kind and can be divided into glycerophospholipids and sphingolipids, wherein the glycerophospholipids include lecithin, cephalin and phosphatidylinositol. Phospholipids are widely present in nature as major components of cell membranes. Phospholipids play a significant role in activating cells, cell signaling, cell withering, maintaining metabolism of living organisms and the balanced secretion of hormones. Phospholipids can also modify biological membranes, dissolve and remove lipid peroxides, and have antioxidant and anti-aging effects. However, in the analysis of biological samples, phospholipids can cause difficulties in the analysis of metabolites or biotin of biological samples, especially when used in conjunction with liquid phase tandem mass spectrometry (LC-MS). This is because of the presence of charged functional groups, negatively charged phosphate groups and positively charged quaternary amino groups in the phospholipid structure, which can lead to interference in quantitative and qualitative analysis, known as matrix effects. In the case of co-elution of phospholipids with the given analyte, which exhibit a form of ion inhibition or mass signal enhancement, phospholipids can additionally greatly shorten the lifetime of the analytical column, which can lead to unpredictable ion inhibition and less reproducible results. It is therefore desirable to remove as much phospholipid as possible during the analysis of biological samples to reduce the effect of phospholipids on the results of the analysis. According to the reports of related documents, most methods for removing phospholipid adopt a mode of diluting a sample by using an organic solvent or liquid-liquid extraction (LLE), but dilution by using the organic solvent is easy to generate adverse changes on the limit of quantitation, and the liquid-liquid extraction mode only uses dichloromethane and tert-butyl methyl type volatile organic solvents to have good phospholipid removal effect, and is complex to operate and large in solvent consumption.

Solid Phase Extraction (SPE) is used as a chromatographic technique, and compared with other sample pretreatment technical methods, the SPE has the advantages of rapidness, convenience, good reproducibility and the like, so that the SPE is rapidly developed and is an essential sample pretreatment means for the analysis specialty. Two common modes of solid phase extraction include, adsorption enrichment of target compounds and impurity rejection by adsorption of impurities, and are widely used, for example, for preconcentration and purification of analytical samples, purification of various chemicals, and removal of toxic or valuable substances from aqueous solutions. SPE is typically performed using a chromatography column containing a suitable material or adsorbent. Sorbent materials have been developed that can interact with analytes by hydrophobic, ion exchange, chelation, adsorption, and other mechanisms to bind and purify analytes from fluids. Therefore, the solid phase extraction technology has great application potential for removing phospholipids in biological samples in the analysis process. The solid phase extraction filler is used as a core device in the solid phase extraction technology, and the technical key is to select the filler with specific adsorption to phospholipid. The fillers for adsorbing phospholipids reported in the literature at present are basically silica gel fillers modified by metal ions or metal oxides, and the disclosure numbers are as follows: chinese patents of CN107640783A and CN111992154A and research literature of yaoka (sp.j.1096.2013.21294) respectively use materials modified by zinc oxide, titanium oxide and zirconium oxide to adsorb phospholipids. In addition, some documents report that cyclodextrin is used to adsorb phospholipids, such as Lajos Szente et al (Struct Chem (2017)28:479-492) utilize the structure and cavity effect of cyclodextrin molecules to adsorb phospholipids. However, the method has the disadvantages of strict reaction conditions, long reaction steps, high requirements on operators, no contribution to scale-up production, heavy metal pollution in the production process and the like.

Disclosure of Invention

The invention aims to solve the technical problem of providing an organic-inorganic hybrid material and a preparation method and application thereof, wherein the hybrid material is a hybrid material of an organic phase and an inorganic phase and can be used for removing phospholipids, lipids and proteins in a matrix; the hybrid material has simple preparation process and good phospholipid removing effect, and can be applied to the application of compounds such as beta receptor agonist, quinolone, antibiotic and the like in a biological matrix sample.

The invention adopts the technical scheme that the organic-inorganic hybrid material is prepared by taking divinyl benzene as a reaction monomer, adding a functional monomer, an organic silicon source, a pore-forming agent and an initiator, and performing copolymerization condensation reaction.

Further, the reaction monomer also comprises N-vinyl pyrrolidone and/or styrene, and the mass ratio of the N-vinyl pyrrolidone or the styrene to the divinylbenzene is 0: 2-1: 45.

further, the functional monomer is vinyl silane or methacryloxypropyl trimethoxy silane, and the mass ratio of the functional monomer to divinylbenzene is 1: 1-1: 12.

further, the organic silicon source is methyl orthosilicate or ethyl orthosilicate, and the mass ratio of the organic silicon source to the divinylbenzene is 1: 4-1: 22.

further, the pore-forming agent is one or more of toluene, mesitylene, n-heptane, n-butanol and liquid paraffin, and the mass ratio of the pore-forming agent to divinylbenzene is 1: 1-45: 1.

further, the initiator is azobisisobutyronitrile or dibenzoyl peroxide.

The invention also provides a preparation method of the organic-inorganic hybrid material for solving the technical problems, wherein the preparation method comprises the following steps: s1) adding a dispersant into a reaction vessel filled with a reaction solvent, and adding divinylbenzene as a reaction monomer after the dispersant is completely dissolved; s2) adding a functional monomer, an organic silicon source, a pore-forming agent and an initiator; s3) reacting at the temperature of 45-95 ℃, and stopping after reacting for 9-30 h; s4) carrying out suction filtration, washing and drying on the obtained product to obtain the organic-inorganic hybrid material.

Further, the reaction solvent is deionized water or an ethanol/deionized water mixed solution; the volume ratio of the ethanol to the deionized water is 1: 3-1: 15.

Further, the dispersing agent is one or more of polyvinylpyrrolidone, hydroxypropyl methylcellulose and polyvinyl alcohol, and the mass ratio of the dispersing agent to divinylbenzene is 1: 10-1: 85.

further, the organic-inorganic hybrid material is used as a solid phase extracting agent for removing phospholipid components in milk, soymilk or egg white matrixes.

Compared with the prior art, the invention has the following beneficial effects: the organic-inorganic hybrid material provided by the invention is a new organic-inorganic hybrid material obtained by copolymerization, condensation and modification of organic-inorganic monomers, and the new material is used as a solid phase extraction filler, and the interaction of polar and nonpolar parts of a phospholipid molecular structure is utilized to achieve the effect of removing more than 99.99% of phospholipid, and the organic-inorganic hybrid material has the advantages of simple operation in the post-treatment process, direct column passing after sample treatment, small solvent consumption, environmental protection and the like.

Drawings

FIG. 1 is a LC-MS spectrum of total phospholipids in a blank sample according to the present invention;

FIG. 2 is a LC-MS spectrum of total phospholipids treated by an adsorption phospholipid solid phase extraction column according to the present invention;

FIG. 3 is a scanning electron microscope characterization chart of examples 1, 2, 3 and 4 of the present invention.

Detailed Description

The invention is further described below with reference to the figures and examples.

The technical scheme of the invention is to provide a novel organic-inorganic hybrid material and a preparation method and application thereof, wherein a reaction monomer, a functional monomer and an organic silicon source monomer are subjected to copolymerization condensation to obtain the novel organic-inorganic hybrid material.

The invention adopts a suspension polymerization method, styrene or N-vinyl pyrrolidone is taken as a reaction monomer to be dissolved in water, a dispersing agent is added, divinylbenzene is taken as a cross-linking agent, toluene, mesitylene or N-butyl alcohol and the like are taken as pore-foaming agents, methyl orthosilicate or ethyl orthosilicate is added as an organic silicon source, vinyl silane or methacryloxypropyl trimethoxysilane is taken as a functional monomer, and the composite material containing an organic phase and an inorganic phase is obtained after copolymerization and condensation. Further, after mechanical stirring is started, polyvinylpyrrolidone is added into a reaction container filled with a reaction solvent, after the polyvinylpyrrolidone is completely dissolved, reaction monomers such as divinylbenzene, ethyl orthosilicate, n-butanol and azobisisobutyronitrile are added, the reaction is stopped after the reaction is carried out for a period of time, and the obtained product is subjected to suction filtration, washing and drying to obtain the novel material.

Further, the reaction solvent is deionized water or an ethanol/deionized water mixed solution (the volume ratio of ethanol to deionized water is 1: 3-1: 15).

Further, the dispersing agent is one or more of polyvinylpyrrolidone, hydroxypropyl methylcellulose and polyvinyl alcohol, and the mass ratio of the dispersing agent to the polymerization monomer divinylbenzene is 1: 10-1: 85.

further, the reaction monomer is one or two of N-vinyl pyrrolidone, styrene and divinylbenzene, and the divinylbenzene is necessarily selected. Wherein the mass ratio of N-vinyl pyrrolidone, styrene and divinylbenzene is 0: 2-1: 45.

further, the functional monomer is one or two of vinyl silane and methacryloxypropyl trimethoxy silane, and the mass ratio of the total addition amount of the functional monomer to the divinylbenzene is 1: 1-1: 12.

further, the organic silicon source is one or two of methyl orthosilicate or ethyl orthosilicate, and the mass ratio of the total adding amount of the organic silicon source to the divinylbenzene is 1: 1-22: 1.

further, the pore-forming agent is one or more of toluene, mesitylene, n-heptane, n-butanol and liquid paraffin, preferably n-heptane and toluene, wherein the mass ratio of the total addition amount of the pore-forming agent to the divinylbenzene is 1: 1-45: 1.

further, the initiator is azobisisobutyronitrile or dibenzoyl peroxide.

Further, the reaction temperature is 45-95 ℃, the reaction time is 9-30 h, and the mechanical stirring speed is 250-700 r/min.

Further, after suction filtration, washing the obtained product with tetrahydrofuran for 1-5 times, then washing the product with methanol or ethanol for 1-5 times, naturally drying the product for 10-30 hours, and then drying the product in vacuum at the drying temperature of 40-90 ℃ for 10-65 hours.

The technical scheme of the invention provides a novel organic-inorganic hybrid material which can be used as a solid phase extraction column filler for adsorbing phospholipid, and the filler is obtained according to the preparation method, and the removal rate of the total phospholipid in phospholipid mixture such as phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and the like in milk reaches 99.99%.

The technical scheme of the invention also comprises that the filler is used as a solid phase extraction column filler for adsorbing phospholipid, lipid impurities can be removed, and the filler can be applied to animal residues and the like.

Compared with the prior art, the invention has the following advantages:

(1) the preparation process of the solid phase extraction filler for adsorbing phospholipid is simple, the raw materials have low toxicity, and the process route is short.

(2) The solid phase extraction filler for adsorbing phospholipid can effectively remove phospholipid components in matrixes such as milk, soymilk, egg white and the like without activation in the pretreatment, the total removal rate of specific phospholipid reaches 99.99 percent, the operation is simple and convenient, the solvent consumption is low, the time consumption is short, and the purification effect is good.

(3) The filler can be applied to the removal of various animal residues, lipids and pigments in a biological matrix sample. If the method is applied to the detection of compounds such as beta-receptor agonist, quinolone, antibiotic and the like, the recovery rate of the added standard sample is not influenced in the purification process, the influence of impurity interferents such as phospholipid and the like can be effectively reduced, the analysis sensitivity of an instrument is improved, and the service life of the instrument is prolonged.

(4) The filler of the invention has good stability and good reproducibility, and is easy for mass production.

The following examples are solid phase extraction column packing materials prepared by the method of the present invention for adsorbing phospholipids and used in dephosphatation and veterinary applications.

Example 1

Adding 1g of polyvinylpyrrolidone into a 500mL three-neck flask filled with 200mL of water, stirring at a mechanical stirring speed of 450 rpm for half an hour until the dispersing agent is completely dissolved; a mixed solution of 15g of divinylbenzene, 1.5g of azobisisobutyronitrile, 9.8g of liquid paraffin, 15g of ethyl orthosilicate and 16g of toluene is added in sequence, the mixture is stirred for half an hour, then the temperature is raised to 70 ℃, and the reaction is stopped after 12 hours of reaction. The product was washed 2 times with 90mL of tetrahydrofuran each time and 2 times with 90mL of methanol each time. The filter cake was air dried naturally for 12h and then vacuum dried at 60 ℃ for 12h to complete the filler preparation of example-1.

Example 2

Adding 1g of polyvinylpyrrolidone into a 500mL three-neck flask filled with 200mL of water, stirring at a mechanical stirring speed of 450 rpm for half an hour until the dispersing agent is completely dissolved; a mixed solution of 15g of divinylbenzene, 8g of styrene, 1.5g of dibenzoyl peroxide, 9.8g of liquid paraffin, 35g of vinyl silane, 60g of ethyl orthosilicate and 16g of n-heptane is sequentially added, the mixture is continuously stirred for half an hour, the temperature is raised to 70 ℃, and the reaction is stopped after 12 hours of reaction. The product was washed 2 times with 90mL of tetrahydrofuran each time and 2 times with 90mL of methanol each time. The filter cake was air dried naturally for 12h and then vacuum dried at 60 ℃ for 12h to complete the filler preparation of example-3.

Example 3

Adding 1.5g of hydroxypropyl methyl cellulose into a 500mL three-neck flask filled with 200mL of water, stirring at the mechanical stirring speed of 450 rpm for half an hour until the dispersing agent is completely dissolved; a mixed solution of 15g of divinylbenzene, 1.5g of azobisisobutyronitrile, 9.8g of liquid paraffin, 35g of methacryloxypropyltrimethoxysilane, 15g of ethyl orthosilicate and 45g of toluene was added in this order, and after stirring for half an hour, the temperature was raised to 70 ℃ to react for 12 hours, and then the reaction was stopped. The product was washed 2 times with 90mL of tetrahydrofuran each time and 2 times with 90mL of methanol each time. The filter cake was air dried naturally for 12h and then vacuum dried at 60 ℃ for 12h to complete the filler preparation of example-5.

Example 4

Adding 1g of polyvinylpyrrolidone into a 500mL three-neck flask filled with 200mL of water, stirring at a mechanical stirring speed of 450 rpm for half an hour until the dispersing agent is completely dissolved; a mixed solution of 40g of divinylbenzene, 1.5g of dibenzoyl peroxide, 9.8g of liquid paraffin, 35g of vinyl silane, 60g of ethyl orthosilicate, 45g of toluene and 15g of n-heptane is sequentially added, the mixture is continuously stirred for half an hour, then the temperature is raised to 70 ℃, and the reaction is stopped after 12 hours of reaction. The product was washed 2 times with 90mL of tetrahydrofuran each time and 2 times with 90mL of methanol each time. The filter cake was air dried naturally for 12h and then vacuum dried at 60 ℃ for 12h to complete the filler preparation of example-6.

Fig. 3 is a scanning electron microscope characterization chart of the synthetic filler of

embodiments

1, 2, 3 and 4 of the present invention, which characterizes that the surface morphology of the synthetic filler is smooth and the spherical structure is stable. Compared with the amorphous filler, the spherical filler has larger adsorption capacity, and the flow rate of the filled solid phase extraction column is faster.

Test example 1

The solid phase extraction column packing prepared in example 1, example 2, example 3 and example 4 was packed with a capacity of 3mL and 60mg of packing per column. Taking a commercial dephosphorizing lipid SPE small column with a certain international known brand as a standard, taking a mixture of phosphatidyl choline, sphingomyelin and phosphatidyl ethanolamine contained in milk as a target, detecting the sum of peak areas of specific parent ion/daughter ion pairs of phospholipid before and after passing through the column by liquid phase mass spectrometry, calculating the removal rate, and investigating the dephosphorizing lipid performance of the filler, wherein the formula is as follows:

removal rate (1- (A/A)₀))×100％

A: the sum of the peak areas of the specific parent ion/daughter ion pairs of all phospholipid mixtures after column chromatography;

A₀: sum of peak areas of specific parent/daughter ion pairs of all phospholipid mixtures before column passage.

The specific operation steps are as follows:

s1, extraction: adding 4mL of 0.2% acetonitrile formate into milk, carrying out vortex oscillation for 1min, centrifuging at 10000r/min for 5min, and collecting supernatant;

s2, loading: adding the supernatant into an SPE small column, and collecting a sample liquid;

s3, constant volume: after nitrogen blowing, 1mL of acetonitrile-0.1% formic acid solution (V/V ═ 1:1) was added to a volume of 1mL for injection.

In this test example, the detection apparatus is Perkinelmer. Instrument reference conditions: mass spectrum: a positive ion scanning mode; source temperature: 110 ℃; the temperature of the desolvation: 350 ℃; detection mode: multiple reactions were monitored, and the monitoring conditions are shown in table 1. Liquid phase conditions: c18 column with length of 100mm, inner diameter of 2.1mm, and particle diameter of 1.8 μm; mobile phase: a: 5mmol ammonium acetate solution B acetonitrile; flow rate: 0.4 mL/min; column temperature: 30 ℃; sample introduction amount: 5 μ L, gradient elution program see Table 1.

TABLE 1 gradient elution procedure

Time/min	Mobile phase A/%)	Mobile phase B/%)
			1.50	95	5
6.00	40	60
			6.10	95	5
8.00	95	5

The phospholipid removal rates for example 1, example 2, example 3, example 4 and commercial fillers are shown in table 2. As can be seen from the data in table 2 in conjunction with fig. 1 and 2, the phospholipid content in the upper liquid without passing through the column is high, the phospholipid in the upper liquid after passing through the column is adsorbed and removed by the filler, and the removal efficiency of the filler prepared by the present invention to phospholipid is above 90%, wherein the removal rate of example 4 is 99.99% which is slightly higher than that of commercial phospholipid-removed filler (99.97%), which indicates that the filler prepared by the present invention can effectively remove phospholipid mixtures such as phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, etc. contained in milk.

TABLE 2 Filler-to-phospholipid removal

Test example 2

The solid phase extraction column fillers prepared in example 1, example 2, example 3 and example 4 are loaded into a column, the capacity of the solid phase extraction column is 3mL, each small column is loaded with 60mg of the filler, the receptor agonist, the quinolone and the antibiotic are added into 1mL of milk in a mixed standard mode, a standard adding and recycling mode is adopted, a column sample is not used as a control, whether the fillers can remove phospholipid interference in the veterinary drug detection process of an actual sample is examined, and the specific operation steps are as follows:

s1, adding a mark: preparing a receptor stimulant with the concentration of 50ppb, quinolone and antibiotic mixed standard, and adding 1mL of milk;

s2, extraction: adding 4mL of 0.2% acetonitrile formate into milk, carrying out vortex oscillation for 1min, centrifuging at 10000r/min for 5min, and collecting supernatant;

s3, loading: adding the supernatant into an SPE small column, and collecting a sample liquid;

s4, constant volume: after nitrogen blowing, 1mL of acetonitrile-0.1% formic acid solution (V/V ═ 1:1) was added to a volume of 1mL for injection.

(comparative example is obtained after the supernatant in S2 is subjected to nitrogen-blowing constant volume, and the supernatant is not subjected to a solid-phase extraction column)

In this test example, the detection apparatus is Perkinelmer. Instrument reference conditions: mass spectrum: a positive ion scanning mode; source temperature: 110 ℃; the temperature of the desolvation: 350 ℃; detection mode: multiple reactions were monitored, and the monitoring conditions are shown in table 3. Liquid phase conditions: c18 column with length of 100mm, inner diameter of 2.1mm, and particle diameter of 1.8 μm; mobile phase: a: 5mmol ammonium acetate solution B acetonitrile; flow rate: 0.4 mL/min; column temperature: 30 ℃; sample introduction amount: 5 μ L, gradient elution program see Table 1.

The recovery of 21 animal residues such as receptor agonists, quinolones and antibiotics from the adsorbed phospholipid solid phase extraction packing of example 1, example 2, example 3 and example 4 and comparative data for commercial packing are shown in table 4. The normalized recovery of examples 1, 2, 3 and 4 was not abnormal by comparison with the control without column, wherein the recovery of 21 targets was greater than 80% for the filler prepared in example 4, which was substantially consistent with commercial filler performance. The filler prepared by the method can remove the interference of phospholipid in the veterinary residue detection process, basically has no influence on the recovery rate of the target object, and can be applied to the removal of phospholipid in the veterinary residue detection process in an actual sample.

TABLE 3 multiple response monitoring conditions for receptor agonists, quinolones and antibiotics

(Note: ions plus "+" for quantitation)

TABLE 4 recovery of filler to receptor agonist, quinolone, and antibiotic

Although the present invention has been described with respect to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. An organic-inorganic hybrid material is characterized in that divinylbenzene is taken as a reaction monomer, a functional monomer, an organic silicon source, a pore-forming agent and an initiator are added, and copolymerization condensation reaction is carried out to obtain the organic-inorganic hybrid material.

2. The organic-inorganic hybrid material according to claim 1, wherein the reaction monomer further comprises N-vinylpyrrolidone and/or styrene, and the mass ratio of N-vinylpyrrolidone or styrene to divinylbenzene is 0: 2-1: 45.

3. the organic-inorganic hybrid material according to claim 1, wherein the functional monomer is vinylsilane or methacryloxypropyltrimethoxysilane, and the mass ratio of the functional monomer to divinylbenzene is 1: 1-1: 12.

4. the organic-inorganic hybrid material according to claim 1, wherein the organic silicon source is methyl orthosilicate or ethyl orthosilicate, and the mass ratio of the organic silicon source to divinylbenzene is 1: 4-1: 22.

5. the organic-inorganic hybrid material according to claim 1, wherein the porogen is one or more of toluene, mesitylene, n-heptane, n-butanol and liquid paraffin, and the mass ratio of the porogen to divinylbenzene is 1: 1-45: 1.

6. the organic-inorganic hybrid material according to claim 1, wherein the initiator is azobisisobutyronitrile or dibenzoyl peroxide.

7. A method for preparing an organic-inorganic hybrid material according to any one of claims 1 to 6, comprising the steps of:

s1) adding a dispersant into a reaction vessel filled with a reaction solvent, and adding divinylbenzene as a reaction monomer after the dispersant is completely dissolved;

s2) adding a functional monomer, an organic silicon source, a pore-forming agent and an initiator;

s3) reacting at the temperature of 45-95 ℃, and stopping after reacting for 9-30 h;

s4) carrying out suction filtration, washing and drying on the obtained product to obtain the organic-inorganic hybrid material.

8. The method for preparing an organic-inorganic hybrid material according to claim 7, wherein the reaction solvent is deionized water or an ethanol/deionized water mixture; the volume ratio of the ethanol to the deionized water is 1: 3-1: 15.

9. The method for preparing an organic-inorganic hybrid material according to claim 7, wherein the dispersant is one or more of polyvinylpyrrolidone, hydroxypropylmethylcellulose and polyvinyl alcohol, and the mass ratio of the dispersant to divinylbenzene is 1: 10-1: 85.

10. use of an organic-inorganic hybrid material according to any one of claims 1 to 6 as a solid phase extractant for removing phospholipid components from a milk, soy milk or egg white matrix.