WO2021098075A1 - Phenylboronic acid solid phase extraction column filling and preparation method therefor - Google Patents

Abstract

Disclosed are a phenylboronic acid solid phase extraction column filling and a preparation method therefor. The preparation method comprises the following steps: subjecting silica gel and an organosilane to a one step reaction for bonding the organosilane on the surface of the silica gel matrix, and then reacting same with a phenylboronic acid monomer. The organosilane is an aminosilane and the phenylboronic acid monomer is 4-carboxyphenylboronic acid. According to the phenylboronic acid solid phase extraction column filling provided by the present invention and the preparation method therefor, the filling is silica gel particles bonded with a phenylboronic acid group and can be used to detect the amount of ribavirin residue in an animal source food; and the filling has a simple preparation process, a short reaction time, mild conditions, and a high yield.

Description

Packing for phenylboric acid solid phase extraction column and preparation method thereof Technical field

The invention relates to a filler of a silica gel matrix solid phase extraction column and a preparation method thereof, in particular to a filler of a phenylboronic acid solid phase extraction column, a preparation method and application thereof.

Background technique

Phenylboronic acid (PBA) is a unique silica-based SPE adsorbent that contains phenylboronic acid functional groups that can retain analytes through reversible covalent bonds. This very strong covalent retention mechanism produces high selectivity and purification efficiency. The borate group has a strong affinity for compounds containing a cis-diol structure, such as catechols, nucleic acids, some proteins, carbohydrates and PEG compounds. Amino acids, α-hydroxyamides, and ketone compounds can also be retained.

Duan Yuhui et al. used the "Clickchemistry" method to react alkynyl 3-aminophenylboronic acid with silica gel azide to prepare a new type of solid phase extraction adsorbent for boric acid. This method has cumbersome steps, difficult purification, and poor selectivity (Duan Yuhui, Wei Yinmao. Dopamine Chromatographic Analysis Method Based on New Boric Acid Solid Phase Extraction Column[J]. Chinese Journal of Analytical Chemistry, 2013,41(3):406-411 ).

Cheng Ting et al. used atom transfer radical polymerization (ATRP) technology to polymerize chain-like functional groups on the surface of the attapulgite, and then increased the specific surface area of the material by introducing gold nanoparticles, and finally grafted mercaptophenylboronic acid to the material through the action of gold and sulfhydryl groups. surface. In solid phase extraction, this material requires a complicated and time-consuming centrifugal separation process. Even with a high specific surface area, the limited amount of grafting still limits its adsorption performance (Cheng Ting. Preparation of new phenylboronic acid materials and their use in biological samples Application of [D]. Lanzhou University, 2017).

Lin et al. used a simple one-pot method to prepare monodisperse core-shell magnetic nanoparticles functionalized with phenylboronic acid. Take FeCl ₃ .6H ₂ O, tetramethyloxysilane and 3-(methacryloxy)propyltrimethoxysilane as precursors, 4-vinylbenzeneboronic acid as functional monomer, and ethylene glycol two Methacrylate is a crosslinking agent. This method has serious deficiencies in adsorption performance and recognition selectivity, and cannot be applied to complex biological samples. (Z. Lin et al. RSC Advances, 2012, 2, 5062-5065).

Chinese Patent Document (CN108409767A) provides a method for preparing heterocyclic biphenylboronic acid. The boronating reagent and organolithium reagent are dissolved in a solvent, and then the reaction is carried out. After the reaction is completed, a basic reagent is used to carry out a hydrolysis reaction to obtain a heterocyclic biphenylboronic acid. Phenylboronic acid filler. This method has low yield and unstable reaction conditions, which is not conducive to large-scale production. At present, the PBA fillers prepared in the above-mentioned methods all have technical difficulties such as long reaction time, cumbersome preparation steps, low yield, and difficulty in large-scale production.

technical problem

The purpose of the present invention is to provide a phenylboronic acid solid phase extraction column packing and a preparation method thereof. The packing is silica gel particles bonded with phenylboronic acid groups, which can be used to detect the residual amount of ribavirin in animal-derived foods; and the packing The preparation process is simple, the reaction time is short, the conditions are mild, and the yield is high.

Technical solutions

The technical scheme of the present invention is to provide a method for preparing phenylboronic acid solid phase extraction column packing, which includes the following steps: reacting silica gel and organosilane in one step, bonding the organosilane on the surface of the silica gel matrix, and then reacting with the phenylboronic acid monomer For the reaction, the organosilane is aminosilane, and the phenylboronic acid monomer is 4-carboxyphenylboronic acid, wherein:

The chemical structural formula of the aminosilane is:

.

Further, the silica gel is porous silica gel, and the porous silica gel is ultra-pure porous amorphous silica gel particles with a particle size range of 40-63 μm and a pore diameter of 60 Å.

Furthermore, silica gel and aminosilane reagent are added to the reaction container with organic solvent in sequence, and the reaction is carried out at a certain reaction temperature. The reaction is mechanically stirred. The reaction is stopped after a period of time. The resulting product is filtered, washed, and dried by suction to obtain Silica gel bonded with amino groups.

Further, the organic solvent is selected from toluene, dichloromethane or N,N-dimethylformamide.

Further, the volume ratio of the mass of the silica gel to the aminosilane reagent is 1: (0.1-5) g/mL.

Further, the reaction temperature is 50-100° C., the reaction time is 4-24 h, and the mechanical stirring speed is 200-500 revolutions per minute.

Furthermore, 4-carboxyphenylboronic acid and an activator were added to the reaction vessel containing the organic solvent in sequence, and the reaction was carried out at normal temperature with mechanical stirring. After a period of reaction, the silica gel containing amino groups was added, and the reaction was carried out at normal temperature. The reaction adopts mechanical stirring, and after a period of reaction, the product obtained is filtered, washed, and dried by suction to obtain a phenylboronic acid solid phase extraction column packing bonded with a phenylboronic acid functional group.

Further, the organic solvent is selected from dimethyl sulfoxide or N,N-dimethylformamide.

Further, the activator is dicyclohexylcarbodiimide, N,N'-carbonyldiimidazole, 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine Hydrochloride, N-hydroxysuccinimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

Further, the mass ratio of the amino silica gel to the 4-carboxyphenylboronic acid reagent is 1: (0.1-5).

Further, the reaction time is 4 to 24 hours, and the mechanical stirring speed is 200 to 500 revolutions per minute.

The technical scheme of the present invention also provides a phenylboronic acid solid phase extraction column packing, which is obtained according to the above preparation method. The packing has a particle size of 40~70μm, a pore diameter of 50~70Å, and a pore volume of 0.5~0.7cm ³ /g , The specific surface area is 200~500m ² /g.

The technical scheme of the present invention also includes the application of the above-mentioned filler as the filler of the phenylboronic acid solid phase extraction column in the separation of ribavirin.

Beneficial effect

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

(1) The preparation process of the invention is simple and the yield is high.

(2) The amount of boronic acid groups on the surface of the silica gel of the present invention can be adjusted, and the amount of boronic acid groups on the surface of the silica gel can be adjusted by controlling the amount of phenylboronic acid.

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

(4) The solid phase extraction column packing prepared by the present invention has good selectivity to ribavirin, and can be widely used in the determination of ribavirin residues in animal-derived foods.

Embodiments of the present invention

The present invention will be further described below in conjunction with the embodiments, but it should not be understood as a limitation to the present invention.

Example 1

Add 20 grams of silica gel to a 250 mL three-necked flask containing 150 mL of toluene, control the mechanical stirring speed to 300 rpm, and then add 5 mL of 3-aminopropyltrimethoxysilane reagent, start to heat up to 60°C, and react for 8 hours Then stop the reaction. After that, the product was washed twice with methanol, using 75 mL of methanol each time, and the filter cake was vacuum dried for 12 hours at 60° C., and the preparation of amino silica gel was completed.

Separately add 10 grams of 4-carboxyphenylboronic acid, 10 grams of N-hydroxysuccinimide and 20 grams of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride into 100mL N , N-dimethylformamide in a 250mL three-necked flask, control the mechanical stirring speed to 300 rpm, after 8 hours of reaction at room temperature, add 50 grams of amino silica gel to the bottle, and stop the reaction after another 8 hours of reaction at room temperature . After that, the product was washed twice with methanol, using 75 mL of methanol each time, and the filter cake was vacuum-dried for 12 hours at 60° C., and the preparation of the PBA filler was completed.

Example 2

Add 20 grams of silica gel to a 250 mL three-necked flask containing 150 mL of toluene, control the mechanical stirring speed to 300 rpm, and then add 10 mL of 3-aminopropyltrimethoxysilane reagent, start to heat up to 60°C, and react for 8 hours Then stop the reaction. After that, the product was washed twice with methanol, using 75 mL of methanol each time, and the filter cake was vacuum dried for 12 hours at 60° C., and the preparation of amino silica gel was completed.

Separately add 10 grams of 4-carboxyphenylboronic acid, 10 grams of N-hydroxysuccinimide and 20 grams of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride into 100mL N , N-dimethylformamide in a 250mL three-necked flask, control the mechanical stirring speed to 300 rpm, after 8 hours of reaction at room temperature, add 30 grams of amino silica gel to the bottle, and stop the reaction after another 8 hours of reaction at room temperature . After that, the product was washed twice with methanol, using 75 mL of methanol each time, and the filter cake was vacuum-dried for 12 hours at 60° C., and the preparation of the PBA filler was completed.

Example 3

Add 20 grams of silica gel to a 250 mL three-necked flask containing 150 mL of toluene, control the mechanical stirring speed to 300 rpm, and then add 15 mL of 3-aminopropyltrimethoxysilane reagent, start to heat up to 60°C, and react for 8 hours Then stop the reaction. After that, the product was washed twice with methanol, using 75 mL of methanol each time, and the filter cake was vacuum dried for 12 hours at 60° C., and the preparation of amino silica gel was completed.

Separately add 10 grams of 4-carboxyphenylboronic acid, 10 grams of N-hydroxysuccinimide and 20 grams of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride into 100mL N , N-dimethylformamide in a 250mL three-necked flask, control the mechanical stirring speed to 300 rpm, after 8 hours of reaction at room temperature, add 20 grams of amino silica gel to the bottle, and stop the reaction after another 8 hours of reaction at room temperature. . After that, the product was washed twice with methanol, using 75 mL of methanol each time, and the filter cake was vacuum-dried for 12 hours at 60° C., and the preparation of the PBA filler was completed.

The phenylboronic acid solid phase extraction column packing prepared in the embodiment of the present invention is referred to as PBA packing. Elemental analysis of PBA packing, particle size and BET data are as follows:

[Corrected according to Rule 26 13.03.2020]

Test case 1

Pack the solid phase extraction column fillers prepared in Examples 1 to 3, the capacity of the solid phase extraction column is 3 mL, and each small column is packed with 100 mg filler. The specific operation steps are as follows:

S1, activation balance: 1mL100mmol/L formic acid solution, 3mLpH 8.5 Ammonium acetate buffer solution;

S2, sample loading: 6mL spiked sample solution, collect the sample solution;

S3. Rinse: 3mL pH 8.5 ammonium acetate buffer solution, collect the eluent, and drain;

S4. Elution: 3mL of 100mmol/L formic acid solution, drained.

In this test example, the standard product is ribavirin, the test concentration on the computer is 20 ppb, and the detection instrument is a liquid chromatography-mass spectrometer. The instrument test standard refers to "SN/T4519-2016 Determination of Ribavirin Residues in Foods of Exported Animal Origin. Liquid Chromatography-Mass Spectrometry/Mass Spectrometry". Instrument reference conditions: zwitterionic hydrophilic interaction chromatography column, column length 100mm, inner diameter 3.0mm, particle size 2.7μm; mobile phase: formic acid and ammonium acetate solution; flow rate: 0.4mL/min; sample volume: 10μL.

By comparing with the peak area of the standard liquid spectrum (the detection concentration is 20ppb), the recovery rate of the external standard and internal standard of the filler to ribavirin is calculated. Two parallel samples were tested for each sample. According to the test results in Table 1, the recovery rates of Examples 1, 2, and 3 showed an increasing trend. The internal and external standard recovery rates of the fillers prepared in Examples 2 and 3 were both high For commercial PBA, it shows good application performance.

[Corrected according to Rule 26 13.03.2020]

Test case 2

Take the chicken liver into a tissue masher for homogenization, mix well, put it into a clean container, add formic acid solution, adjust PH=8.5 with ammonia, then add pH=8.5 ammonium acetate buffer solution and mix well, extract the supernatant spare.

Pack the solid phase extraction column fillers prepared in Examples 1 to 3, the capacity of the solid phase extraction column is 3 mL, and each small column is packed with 100 mg filler. The specific operation steps are as follows:

S1, activation balance: 1mL100mmol/L formic acid solution, 3mLpH 8.5 Ammonium acetate buffer solution;

S2, sample loading: 6mL spiked sample solution to be loaded;

S3. Rinse: rinse with 3mL pH 8.5 ammonium acetate buffer solution and drain;

S4. Elution: 1 mL of 100mmol/L formic acid solution, drain it, and collect the eluate.

In this test example, the standard product is ribavirin, the test concentration on the computer is 20 ppb, and the detection instrument is a liquid chromatography-mass spectrometer. The instrument test standard refers to "SN/T4519-2016 Determination of Ribavirin Residues in Foods of Exported Animal Origin. Liquid Chromatography-Mass Spectrometry/Mass Spectrometry". Instrument reference conditions: zwitterionic hydrophilic interaction chromatography column, column length 100mm, inner diameter 3.0mm, particle size 2.7μm; mobile phase: formic acid and ammonium acetate solution; flow rate: 0.4mL/min; sample volume: 10μL.

Two parallel samples are tested for each sample (the detection concentration is 20ppb). Table 2 is the average value of the two parallel samples. According to the test results in Table 2, the recovery rates of Examples 1, 2, and 3 show an increasing trend. The external standard recovery rates of the fillers prepared in Examples 2 and 3 are all higher than those of commercial PBA, while the internal standard recovery rates are similar to it, indicating good application performance.

[Corrected according to Rule 26 13.03.2020]

Although the present invention has been disclosed as above in preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications and improvements without departing from the spirit and scope of the present invention. Therefore, the present invention The scope of protection shall be as defined in the claims.

Claims (10)

1. A preparation method of phenylboronic acid solid phase extraction column packing is characterized in that it comprises the following steps: reacting silica gel and organosilane in one step, bonding the organosilane on the surface of the silica gel matrix, and then reacting with the phenylboronic acid monomer, The organosilane is aminosilane, and the phenylboronic acid monomer is 4-carboxyphenylboronic acid, wherein:

   The chemical structural formula of the aminosilane is:

   

   .
2. The preparation method according to claim 1, wherein the silica gel is porous silica gel, and the porous silica gel is ultra-pure porous amorphous silica gel particles with a particle size range of 40-63 μm and a pore diameter of 60 Å.
3. The preparation method according to claim 1, characterized in that the silica gel and aminosilane reagent are sequentially added into the reaction vessel containing the organic solvent, and the reaction is carried out at a certain reaction temperature, and mechanical stirring is adopted. The reaction is stopped after a period of time. The obtained product is filtered with suction, washed, and dried to obtain silica gel bonded with amino groups.
4. The preparation method according to claim 3, wherein the organic solvent is selected from toluene, dichloromethane or N,N-dimethylformamide.
5. The preparation method according to claim 3, wherein the volume ratio of the mass of the silica gel to the aminosilane reagent is 1: (0.1-5) g/mL.
6. The preparation method according to claim 3, wherein the reaction temperature is 50-100° C., the reaction time is 4-24 h, and the mechanical stirring speed is 200-500 revolutions per minute.
7. The preparation method according to claim 1, characterized in that 4-carboxyphenylboronic acid and activator are sequentially added into a reaction vessel filled with an organic solvent, and reacted at room temperature with mechanical stirring, and then added after a period of reaction. The silica gel containing amino groups is reacted at normal temperature with mechanical stirring. After a period of reaction, the resulting product is suction filtered, washed, and dried to obtain a phenylboronic acid solid phase extraction column packing bonded with phenylboronic acid functional groups.
8. The preparation method according to claim 7, wherein the organic solvent is selected from dimethyl sulfoxide or N,N-dimethylformamide.
9. The preparation method according to claim 7, wherein the activator is dicyclohexylcarbodiimide, N,N'-carbonyldiimidazole, 4-(4,6-dimethoxytriazine- 2-yl)-4-methylmorpholine hydrochloride, N-hydroxysuccinimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
10. The preparation method according to claim 7, wherein the mass ratio of the amino silica gel to the 4-carboxyphenylboronic acid reagent is 1: (0.1-5).

    11. The preparation method according to claim 7, wherein the reaction time is 4-24 h, and the mechanical stirring speed is 200-500 revolutions per minute.

    12. A phenylboronic acid solid phase extraction column packing, characterized in that it is obtained according to the preparation method of any one of claims 1-11, the packing has a particle size of 40-70 μm, a pore diameter of 50-70 Å, and The volume is 0.5~0.7cm ³ /g, and the specific surface area is 200~500m ² /g.

    13. The application of the packing of claim 12 as the packing of phenylboronic acid solid phase extraction column in the separation of ribavirin.