CA3109058C

CA3109058C - Benzeneboronic acid solid-phase extraction column packing and preparation method thereof

Info

Publication number: CA3109058C
Application number: CA3109058A
Authority: CA
Inventors: Yibiao Zou; Xinzhu Yan; Wulian Chen
Original assignee: Anple Laboratory Technologies Shanghai Inc
Current assignee: Anple Laboratory Technologies Shanghai Inc
Priority date: 2019-11-20
Filing date: 2020-02-27
Publication date: 2021-10-26
Anticipated expiration: 2040-02-27
Also published as: JP2022503681A; CA3109058A1; GB2589278A; GB202102631D0; JP7113242B2; GB2589278B

Abstract

The invention discloses a phenylboronic acid solid-phase extraction column packing and a preparation method thereof, and the preparation method includes the steps of:
performing one-step reaction on silica gel and organosilane, bonding organosilane on the surface of a silica gel substrate, and then reacting with a phenylboronic acid monomer, where the organosilane is aminosilane, and the phenylboronic acid monomer is 4-carboxyphenylboronic acid. The invention provides a phenylboronic acid solid-phase extraction column packing and a preparation method thereof, and the packing is silica gel particles bonded with phenylboronic acid groups and can be used for determination of ribavirin residue in foodstuffs of animal origin; and the packing is simple in preparation process, short in reaction time, mild in condition and high in yield.

Description

Benzeneboronic Acid Solid-phase Extraction Column Packing and Preparation Method Thereof Technical Field The invention relates to a silica gel matrix solid-phase extraction column packing and a preparation method thereof, in particular to a phenylboronic acid solid-phase extraction column packing, a preparation method and use thereof.
Background Art Phenylboronic acid (PBA) is a unique silica-based SPE adsorbent containing phenylboronic acid functional groups capable of retaining analytes through reversible covalent bonds. This very strong covalent retention mechanism results in very high selectivity and purification efficiency. Borate groups have strong affinity for compounds containing cis-diol structures, such as catechols, nucleic acids, some proteins, carbohydrates and PEG compounds. Amino acids, a-hydroxyamides, and ketones can also be retained.
Duan Yuhui et al. prepared a novel boric acid solid-phase extraction adsorbent through a reaction of alkynylated 3-aminobenzylboronic acid with azidation silica gel using a "Click chemistry" method. The method is complicated in operation steps, difficult in purification, and poor in selectivity (Duan Yuhui, Wei Yinmao.
Dopamine chromatographic analysis method based on novel boric acid solid-phase extraction column [J]. 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, then increased the specific surface area of the material by introducing gold nanoparticles, and finally grafted mercaptophenylboronic acid to the surface of material through the effect of gold and mercapto groups. 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 (Chengting, Preparation of novel phenylboronic acid material and use thereof in biological samples [D]. Lanzhou University, 2017).
Lin et al. prepared monodisperse core-shell magnetic nanoparticles based on ]
Date Recue/Date Received 2021-02-12 phenylboronic acid functionalization by a simple one-pot method. FeC13.6H20, tetramethyloxysilane and 3-(methacryloxy) propyltrimethoxysilane were used as precursors, 4-vinylphenylboronic acid was used as functional monomer, and ethylene glycol dimethacry late was used as a crosslinking agent. The method has serious shortcomings in adsorption performance and recognition selectivity, and cannot be applied to complex biological samples. (Z. Lin et al. RSC Advances, 2012, 2, 5062-5065).
The Chinese patent document (CN108409767A) provides a preparation method of heterocyclic biphenyl boronic acid, where a bonding reagent and an organolithium reagent are dissolved in a solvent and then reacted, and after the reaction is finished, a basic reagent is used for hydrolysis reaction to obtain a heterocyclic biphenyl boronic acid packing. The method is low in yield, unstable in reaction conditions and not favorable for large-scale production. The PBA packing prepared by the method has the technical difficulties of being long in reaction time, complex in preparation steps, low in yield, and difficulty in large-scale production.
Summary of the Invention The invention aims to provide a phenylboronic acid solid-phase extraction column packing and a preparation method thereof, where the packing is silica gel particles bonded with phenylboronic acid groups and can be used for determination of ribavirin residue in foodstuffs of animal origin; and the packing is simple in preparation process, short in reaction time, mild in condition and high in yield.
The technical scheme of the present invention is to provide a preparation method of a phenylboronic acid solid-phase extraction column packing, including the steps of:
performing one-step reaction on silica gel and organosilane, bonding organosilane on the surface of a silica gel substrate, and then reacting with a phenylboronic acid monomer, where the organosilane is aminosilane, and the phenylboronic acid monomer is 4-carboxyphenylboronic acid, and the aminosilane has a chemical structural formula as follows:
NH, Furthermore, the silica gel is porous silica gel, the porous silica gel is ultra-pure

2 Date Recue/Date Received 2021-02-12 porous amorphous silica gel particles, with a particle size ranging from 40 pm to 63 pm, and a pore size of 60 A.
Furthermore, the silica gel and aminosilane reagent are sequentially added into a reaction vessel filled with an organic solvent, then reacted at a certain reaction temperature under mechanical stirring, the reaction is stopped after a period of time, and the reactant is subjected to suction filtration, washing and drying to obtain the silica gel bonded with amino groups.
Furthermore, the organic solvent is selected from toluene, dichloromethane or N,N-di methy lformami de.
Furthermore, a ratio of the mass of silica gel to the volume of aminosilane reagent is 1:
(0.1-5) g/mL.
Furthermore, the reaction temperature ranges from 50 C to 100 C, the reaction time ranges from 4 h to 24 h, and the mechanical stirring speed ranges from 200 rpm to 500 rpm.
Furthermore, 4-carboxyphenylboronic acid and an activating agent are sequentially added into a reaction vessel filled with an organic solvent, then reacted at room temperature under mechanical stirring, the silica gel containing amino groups is added after a period of reaction, the mixture is reacted at room temperature under mechanical stirring, and after a period of reaction, the reactant is subjected to suction filtration, washing and drying to obtain the phenylboronic acid solid-phase extraction column packing bonded with phenylboronic acid functional groups.
Furthermore, the organic solvent is selected from dimethylsulfoxide or N,N-di methy lformami de.
Furthermore, the activating agent consists of dicy clohexy lcarbo di imi de, N,N'-carbonyldiimidazole, 4-(4,6-dimethoxytri azin-2-y1)-4-methylmorpholine hydrochloride, N-hy droxy succinimi de or 1-(3 -dimethy laminopropy1)-3-ethy lcarbodi imi de hydrochloride.

3 Date Recue/Date Received 2021-02-12 Furthermore, a mass ratio of the amino silica gel to 4-carboxyphenylboronic acid reagent is 1: (0.1-5).
Furthermore, the reaction time ranges from 4 h to 24 h, and the mechanical stirring speed ranges from 200 rpm to 500 rpm.
The technical scheme of the invention also provides a phenylboronic acid solid-phase extraction column packing which is obtained according to the preparation method, where the packing has a particle size of 40 p.m to 70 pm, a pore size of 50 A
to 70 A, a pore volume of 0.5 cm3/g to 0.7 cm3/g, and a specific surface area of 200 m2/g to 500 m2/g.
The technical scheme of the invention also includes use of the above packing as the packing for phenylboronic acid solid-phase extraction column in the separation of ribavirin.
Compared with the prior art, the present invention has the following advantages:
(1) the present invention is simple in preparation process and high in yield;
(2) the amount of boric acid groups on the surface of the silica gel can be regulated, and the amount of boric acid groups on the surface of the silica gel can be regulated by controlling the amount of phenylboronic acid;
(3) the packing of the present invention has good stability, good reproducibility, and is easy for mass production; and

(4) the solid-phase extraction column packing prepared by the present invention has good selectivity for ribavirin, and can be widely used for determination of ribavirin residue in foodstuffs of animal origin.
Detailed Description of the Invention The present invention will be further described below in conjunction with the Examples, but it should not be understood as a limitation to the present invention.

Date Recue/Date Received 2021-02-12 Example 1 20 g of silica gel was added to a 250 mL three-necked flask filled with 150 mL
of toluene, with a mechanical stirring speed controlled to 300 rpm, then 5 mL of 3-aminopropyltrimethoxysilane reagent was added, the temperature was initially raised to 60 C, and the reaction was stopped after 8 h. The reactant was then washed twice with 75 mL of methanol each time, and the filter cake was dried under vacuum for 12 h at 60 C to prepare an amino silica gel.
g of 4-carboxyphenylboronic acid, 10 g of N-hydroxysuccinimide and 20 g of 1-(3 -dimethy laminopropy1)-3-ethy lcarbodi imi de hydrochloride were respectively added into a 250 mL three-necked flask filled with 100 mL of N,N-dimethylformamide, with the mechanical stirring speed controlled to be 300 rpm, then 50 g of amino silica gel was added into the flask after 8 h of reaction at normal temperature, and the reaction was stopped after another 8 h of reaction at room temperature. The reactant was then washed twice with 75 mL of methanol each time, and the filter cake was dried under vacuum for 12 h at 60 C to prepare a PBA
packing.
Example 2 g of silica gel was added to a 250 mL three-necked flask filled with 150 mL of toluene, with a mechanical stirring speed controlled to 300 rpm, then 10 mL of 3-aminopropyltrimethoxysilane reagent was added, the temperature was initially raised to 60 C, and the reaction was stopped after 8 h. The reactant was then washed twice with 75 mL of methanol each time, and the filter cake was dried under vacuum for 12 h at 60 C to prepare an amino silica gel.
10 g of 4-carboxyphenylboronic acid, 10 g of N-hydroxysuccinimide and 20 g of 1-(3 -dimethy laminopropy1)-3-ethy lcarbodi imi de hydrochloride were respectively added into a 250 mL three-necked flask filled with 100 mL of N,N-dimethylformamide, with the mechanical stirring speed controlled to be 300 rpm, then 30 g of amino silica gel was added into the flask after 8 h of reaction at normal temperature, and the reaction was stopped after another 8 h of reaction at room temperature. The reactant was then washed twice with 75 mL of methanol each time, and the filter cake was dried under vacuum for 12 h at 60 C to prepare a PBA
packing.

5 Date Recue/Date Received 2021-02-12 Example 3 20 g of silica gel was added to a 250 mL three-necked flask filled with 150 mL
of toluene, with a mechanical stirring speed controlled to 300 rpm, then 15 mL of 3-aminopropyltrimethoxysilane reagent was added, the temperature was initially raised to 60 C, and the reaction was stopped after 8 h. The reactant was then washed twice with 75 mL of methanol each time, and the filter cake was dried under vacuum for 12 h at 60 C to prepare an amino silica gel.
g of 4-carboxyphenylboronic acid, 10 g of N-hydroxysuccinimide and 20 g of 1-(3 -dimethy laminopropy1)-3-ethy lcarbodi imi de hydrochloride were respectively added into a 250 mL three-necked flask filled with 100 mL of N,N-dimethylformamide, with the mechanical stirring speed controlled to be 300 rpm, then 20 g of amino silica gel was added into the flask after 8 h of reaction at normal temperature, and the reaction was stopped after another 8 h of reaction at room temperature. The reactant was then washed twice with 75 mL of methanol each time, and the filter cake was dried under vacuum for 12 h at 60 C to prepare a PBA
packing.
The phenylboronic acid solid-phase extraction column packing prepared in the Examples of the present invention is referred to as a PBA packing, and elemental analysis, particle size and BET data of the PBA packing are as follows:
Elemental analysis BET
Particle Sample C [%1 H [%1 N [%1 size Specific Pore size Pore surface area A volume myg cm3/g Example 5.99 1.636 1.04 62.286 338.363 63.51 0.537 Example 5.87 1.631 0.99 67.011 319.27 66.29 0.529 Example 5.86 1.53 0.89 68.573 338.739 65.59 0.555

6 Date Recue/Date Received 2021-02-12 Test Example 1 The solid-phase extraction column packing prepared in Examples 1 to 3 was loaded in the column with a capacity of 3 mL, and each small column was loaded with 100 mg of packing; the specific operation steps were as follows:
Si. activation or condition: 1 mL of 100 mmol/L formic acid solution, 3 mL of pH 8.5 ammonium acetate buffer solution;
S2. loading: 6 mL of labeled sample solution to be loaded, collecting the sample solution;
S3. washing: 3 mL of pH 8.5 ammonium acetate buffer solution, collecting washed material, and performing vacuum drying; and S4. elution: 3 mL of 100 mmol/L formic acid solution, performing vacuum drying.
In the test example, the standard was ribavirin, the on-line detection concentration was 20 ppb, and the detection instrument was a liquid chromatography-mass spectrometer. Instrumental test standards referred to "SN/T4519-2016 Determination of ribavirin residues in foodstuffs of animal origin for export-LC-MS/MS
Method".
Instrument reference conditions: zwitterionic hydrophilic interaction chromatography column, column length 100 mm, internal diameter 3.0 mm, particle size 2.7 pm;
mobile phase: formic acid and ammonium acetate solution; flow Rate: 0.4 mL/min;
sample injection amount: 10 pL.
The recovery rates of the external standard and the internal standard of the packing for ribavirin were calculated by comparing with the peak area of the standard liquid spectrum (detection concentrations of 20 ppb). 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, and the recovery rates of the internal standard and external standard of the packings prepared in Examples 2 and 3 were higher than those of commercial PBA, indicating good application performance.

7 Date Recue/Date Received 2021-02-12 Table 1 Recovery Rate of Ribavirin by PBA Packing Recovery Recovery Ribavirin Ribavirin rate by rate by external internal Sample external internal standard peak standard peak standard standard area area method method Elution-1 58 45 Example 1 18.23 87.48 Elution-2 73 59 Elution-1 101 77 Example 2 28.12 95.38 Elution-2 101 70 Elution-1 215 180 Example 3 51.1 94.78 Elution-2 230 198 Commercial Elution-1 87 62 21.02 92.92 PBA Elution-2 64 50 Test Example 2 Chicken liver was homogenized in a tissue masher, followed by fully and unifointly blending, putting into a clean container, adding a formic acid solution, adjusting pH
value to 8.5 by using ammonia water, then adding an ammonium acetate buffer solution with a pH value of 8.5 for unifointly mixing, and the supernatant was extracted for later use.
The solid-phase extraction column packing prepared in Examples 1 to 3 was loaded in the column with a capacity of 3 mL, and each small column was loaded with 100 mg of packing; the specific operation steps were as follows:
51. activation or condition: 1 mL of 100 mmol/L formic acid solution, 3 mL of pH 8.5 ammonium acetate buffer solution;
S2. loading: 6 mL of labeled sample solution to be loaded;
S3. washing: 3 mL of pH 8.5 ammonium acetate buffer solution, performing vacuum drying;
S4. elution: 1 mL of 100 mmol/L formic acid solution, performing vacuum drying,

8 Date Recue/Date Received 2021-02-12 and collecting the eluate.
In the test example, the standard was ribavirin, the on-line detection concentration was 20 ppb, and the detection instrument was a liquid chromatography-mass spectrometer. Instrumental test standards referred to "SN/T4519-2016 Determination of ribavirin residues in foodstuffs of animal origin for export-LC-MS/MS
Method".
Instrument reference conditions: zwitterionic hydrophilic interaction chromatography column, column length 100 mm, internal diameter 3.0 mm, particle size 2.7 pm;
mobile phase: formic acid and ammonium acetate solution; flow Rate: 0.4 mL/min;
sample injection amount: 10 pL.
Two parallel samples (detection concentrations of 20 ppb) were tested for each sample, and the average values of the two parallel samples were shown in Table 2.
According to the test results in Table 2, the recovery rates of Examples 1, 2 and 3 showed an increasing trend. The recovery rates of the external standard of the packings prepared in Examples 2 and 3 were higher than those of commercial PBA, while the internal standard has a similar recovery rate, indicating good application performance.
Table 2 Recovery Rate of Ribavirin in Matrix by PBA Packing Recovery Recovery Ribavirin Ribavirin rate by rate by external internal Sample external internal standard peak standard peak standard standard area area method method Matrix standard 405800 291500 Example 1 Labeled 9.06 89.92 sample 37190 31480 Matrix standard 364400 292100 Example 2 Labeled 39.51 95.61 sample 134700 123500 Matrix standard 287500 247400 Example 3 Labeled 55.76 99.89 sample 160300 141700 Matrix Commercial standard 364400 319000 Labeled 25.47 99.74 sample

9 Date Recue/Date Received 2021-02-12 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 protection scope of the present invention shall be defined by the claims.
Date Recue/Date Received 2021-02-12

Claims

1. A preparation method of a phenylboronic acid solid-phase extraction column packing, characterized by comprising the steps of: performing one-step reaction on silica gel and organosilane, bonding organosilane on the surface of a silica gel substrate, and then reacting with a phenylboronic acid monomer, wherein the organosilane is aminosilane, and the phenylboronic acid monomer is 4-carboxyphenylboronic acid; and the aminosilane has a chemical structural formula as follows:

2. The preparation method according to claim 1, characterized in that the silica gel is porous silica gel, the porous silica gel is ultra-pure porous amorphous silica gel particles, with a particle size ranging from 40 um to 63 um, and a pore size of 60 A.

3. The preparation method according to claim 1, characterized in that the silica gel and aminosilane reagent are sequentially added into a reaction vessel filled with an organic solvent, then reacted at a certain reaction temperature under mechanical stirring, the reaction is stopped after a period of time, and the reactant is subjected to suction filtration, washing and drying to obtain the silica gel bonded with amino groups.

4. The preparation method according to claim 3, characterized in that the organic solvent is selected from the group consisting of toluene, dichloromethane and N,N-di methy lformami de.

5. The preparation method according to claim 3, characterized in that a ratio of the mass of silica gel to the volume of aminosilane reagent is 1: (0.1-5) g/mL.

6. The preparation method according to claim 3, characterized in that the reaction temperature ranges from 50 C to 100 C, the reaction time ranges from 4 h to 24 h, and the mechanical stirring speed ranges from 200 rpm to 500 rpm.

7. The preparation method according to claim 1, characterized in that 4-carboxyphenylboronic acid and an activating agent are sequentially added into a reaction vessel filled with an organic solvent, then reacted at room temperature under mechanical stirring, the silica gel containing amino groups is added after a period of reaction, the mixture is reacted at room temperature under mechanical stirring, and after a period of reaction, the reactant is subjected to suction filtration, washing and drying to obtain the phenylboronic acid solid-phase extraction column packing bonded with phenylboronic acid functional groups.

8. The preparation method according to claim 7, characterized in that the organic solvent is selected from the group consisting of dimethylsulfoxide and N,N-di methy lformami de.

9. The preparation method according to claim 7, characterized in that the activating agent is dicyclohexylcarbodiimide, N,N'-carbonyldiimidazole, 4-(4,6-dimethoxy tri azi n-2-y1)-4-methy lmorpho line hy drochl ori de, N-hydroxysuccinimide or 1-(3-di methy laminopropy1)-3-ethy lcarbo di imi de hy drochloride.

10. The preparation method according to claim 7, characterized in that a mass ratio of the amino silica gel to 4-carboxyphenylboronic acid reagent is 1: (0.1-5).

11. The preparation method according to claim 7, characterized in that the reaction time ranges from 4 h to 24 h, and the mechanical stirring speed ranges from 200 rpm to 500 rpm.

12. A phenylboronic acid solid-phase extraction column packing obtained according to the preparation method of any one of claims 1 to 11, characterized in that the packing has a particle size of 40 pm to 70 pm, a pore size of 50A to 70A, a pore volume of 0.5 cm3/g to 0.7 cm3/g, and a specific surface area of 200 m2/g to 500 m2/g.

13. Use of the packing according to claim 12 as the packing for phenylboronic acid solid-phase extraction column in the separation of ribavirin.