CN115453031B - Preparation method and application of solid phase extraction column filler special for ribavirin - Google Patents
Preparation method and application of solid phase extraction column filler special for ribavirin Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a solid phase extraction column filler special for ribavirin, wherein the preparation method comprises the following steps: reacting phenylboronic acid monomer with an organosilane reagent to obtain an integrated phenylboronic acid silane reagent; and (3) modifying the integrated phenylboronic acid silane reagent to the surface of spherical silica gel, and reacting off residual silicon hydroxyl on the surface of the silica sphere by using a tail sealing reagent to obtain the ribavirin Lin Zhuanyong solid-phase extraction column filler. The preparation process is simple, the catalyst is used for synergistic catalysis, and the grafting efficiency is high; the bonding amount of the boric acid groups on the surface of the spherical silica gel is easy to regulate and control, and the spherical silica gel is suitable for large-scale production. When the ribavirin recovery rate test is carried out on animal-derived foods, the data show that the recovery rate is up to 90.6%, and the application performance meets the requirements of commercial PBA fillers.
Description
Technical Field
The invention relates to a preparation method and application of a silica gel matrix solid phase extraction column filler, in particular to a preparation method and application of a ribavirin special solid phase extraction column filler.
Background
Ribavirin (chemical name is 1-beta-D-ribofuranosyl-1H-1, 2, 4-triazole-3-carboxamide) is a broad-spectrum powerful antiviral agent with an ortho-cis diol structure, and has certain genetic, reproductive toxicity and carcinogenicity due to great toxic and side effects, and if livestock misuse produces drug residues, the drug residues enter human bodies to produce health hazards. At present, ribavirin is forbidden in animal husbandry, but some illegal merchants reduce the breeding cost, improve the curative effect of treating animal viral infectious diseases (chicken pox, infectious laryngotracheitis, and the like), and still excessively add the antibiotics illegally to seriously damage the food safety.
The boric acid group of phenylboronic acid can form a stable five-membered ring complex with cis-dihydroxyl compound, and reversible covalent bond formation or dissociation can be generated under alkaline/acidic conditions. This particular property can be applied to the enrichment and separation of cis-dihydroxy compounds (e.g., ribavirin, saccharides, nucleosides) in a solid phase pre-extraction treatment. However, most phenylboronic acid functionalized materials have poor selectivity and low adsorption capacity, and cannot effectively enrich cis-dihydroxy compounds in samples.
The alkynylated 3-aminophenylboronic acid reacts with the azidated silica gel by using a click chemistry method such as section glory, etc., so as to prepare the novel boric acid solid-phase extraction adsorbent. The method has complicated steps, difficult purification, high toxicity and poor selectivity of sodium azide (section glory, wei Yin metallocene. Dopamine chromatographic analysis method based on novel boric acid solid phase extraction column [ J ]. Analytical chemistry 2013,41 (3): 406-411). The phenylboronic acid polymer based on the ring-opening polymerization of amino epoxy group disclosed in Chinese patent CN109337080B and a preparation method and application thereof are characterized in that m-aminophenylboronic acid, tri (4-hydroxyphenyl) methane triglycidyl ether, hexamethylenediamine and amino magnetic spheres are dispersed in a mixed solution of dimethyl sulfoxide and polyethylene glycol 200 for reaction, so that the phenylboronic acid polymer with specific adsorption capacity on a hydrophobic nitrogen-containing compound is prepared, and a large amount of dimethyl sulfoxide with higher activity is used as a reaction solvent and a washing solvent in the reaction, so that a large amount of corrosive gas is generated, environmental pollution and corrosion of experimental equipment are caused, and the polymer is unfavorable for green environmental protection and large-scale production.
From the above, the existing processes for preparing phenylboronic acid filler have the technical difficulties of long reaction time, complicated preparation steps, low adsorption capacity, poor selectivity, adoption of corrosive solvents, difficulty in mass production and the like.
Disclosure of Invention
The invention aims to provide a preparation method and application of a solid phase extraction column filler special for ribavirin. The filler is bonded to the surface of spherical silica gel through preparing integral phenylboronic acid silane, and shields residual silicon hydroxyl on the silica gel, so that the residual silicon hydroxyl and certain impurities are prevented from being adsorbed, and the recovery rate of a target object is reduced; the preparation of the integrated silane enables the functional group on the surface of the silicon sphere to be easy to regulate and control, and the filler has high adsorption capacity and high selectivity.
The technical scheme of the invention is as follows: a preparation method of a solid phase extraction column filler special for ribavirin comprises the following steps:
s1) preparation of an integrated phenylboronic acid silane reagent: starting mechanical stirring for 150-500 r/min, adding an organic solvent, adding phenylboronic acid monomer and organosilane into a reaction vessel for reaction, opening condensed water, heating to 70-105 ℃, adding a catalyst, reacting for 4-10 hours, washing the product for 2-3 times respectively by suction filtration, tetrahydrofuran and ethanol, and drying at 40-80 ℃ for 6-20 hours to obtain an integrated phenylboronic acid silane reagent;
s2) modifying phenylboronic acid silane and a tail sealing reagent to the surface of spherical silica gel: starting mechanical stirring for 200-450 r/min, adding an organic solvent, adding spherical silica gel and the integrated phenylboronic acid silane reagent prepared in the step S1, opening condensed water, heating to 70-105 ℃, reacting for 4-15 hours, continuously adding a tail sealing reagent, reacting for 3-6 hours, and stopping; and (3) washing the obtained product for 2-3 times by suction filtration, tetrahydrofuran and ethanol respectively, wherein the drying temperature is 40-80 ℃ and the drying time is 6-20 h, so as to obtain the silica gel bonded with phenylboronic acid functional groups.
Further, the organic solvent in the step S1 is a mixed solution of ethanol and ethyl acetate or a mixed solution of tetrahydrofuran and petroleum ether, the volume ratio of ethanol to ethyl acetate is 1:4, and the volume ratio of tetrahydrofuran to petroleum ether is 2:3, the volume mass ratio of the organic solvent to the phenylboronic acid monomer is 5-20 mL:1g.
Further, the phenylboronic acid monomer in the step S1 is 4-aminophenylboronic acid.
Further, the organosilane in the step S1 is selected from any one of trimethoxy [2- (7-oxabicyclo [4.1.0] hept-3-yl) ethyl ] silane and triethoxy- [2- (7-oxazolicyclo [4.1.0] heptane-3-yl) ethyl ] silane, and the mass ratio of the organosilane reagent to phenylboronic acid monomer is 4.5-10: 1.
further, the catalyst in the step S1 is any one or two of zinc perchlorate hexahydrate and stannous isooctanoate, and the mass ratio of the zinc perchlorate hexahydrate to the stannous isooctanoate is 0.5: 1-3, wherein the mass ratio of the catalyst to the phenylboronic acid monomer is 1:10 to 39.
Further, the spherical silica gel in the step S2 is ultra-pure porous spherical silica gel, the particle size range is 50-75 mu m, the specific surface area is 350-550 m < 2 >/g, and the pore diameter is the same as that of the porous spherical silica gel
Further, in the step S2, the organic solvent is any one of dichloroethane, petroleum ether and tetrahydrofuran, and the volume mass ratio of the organic solvent to the spherical silica gel is 3-20 mL:1g.
Further, in the step S2, the mass ratio of the integral phenylboronic acid silane reagent to the spherical silica gel is 0.2-5: 1.
further, in the step S2, the tail sealing reagent is trimethoxy silane, and the mass ratio of the tail sealing reagent to the spherical silica gel is 1-5: 28.
the filler provided by the invention is used as the filler of the solid-phase extraction column of ribavirin Lin Zhuanyong in the detection of ribavirin in animal-derived foods, the recovery rate in the detection of ribavirin is up to 90.6%, which is higher than the recovery rate of ribavirin Lin Zhuanyong columns of the same type in the market by 82.7%, and a new choice is provided for the detection of ribavirin in animal-derived foods.
Compared with the prior art, the invention has the following beneficial effects: according to the invention, spherical silica gel is adopted to replace amorphous silica gel, so that the bonding uniformity of phenylboronic acid functional groups can be improved, the adsorption capacity of the filler is improved, and the filler has high selectivity to ribavirin Lin Mubiao; the bonding amount of the phenylboronic acid group can also be regulated and controlled by the addition amount of the integrated phenylboronic acid silane reagent; the residual silicon hydroxyl on the surface of the spherical silica gel is shielded, so that the adsorption of impurities on the filler can be reduced, and the detection recovery rate of trace target substances can be improved; the preparation method disclosed by the invention is simple in preparation process, good in stability and reproducibility, easy for large-scale production, and better in enrichment adsorption effect on ribavirin than that of ribavirin Lin Zhuanyong columns of the same type on the market, and can be widely applied to detection of ribavirin in animal-derived foods.
Drawings
FIG. 1 is a scanning electron microscope image of a ribavirin Lin Zhuanyong column packing prepared in accordance with the present invention;
fig. 2 is a liquid chromatogram of ribavirin standard;
FIG. 3 is a liquid chromatogram of example 2 of the present invention for ribavirin;
fig. 4 is a liquid chromatogram of a commercial PBA filler versus ribavirin.
Detailed Description
The invention is further described below in connection with examples, which should not be construed as limiting the invention.
The invention provides a preparation method of a solid phase extraction column filler special for ribavirin, which comprises the following steps:
s1) preparation of integral phenylboronic acid silane reagent: starting mechanical stirring for 150-500 r/min, adding an organic solvent, adding phenylboronic acid monomer and organosilane into a reaction vessel for reaction, opening condensed water, heating to 70-105 ℃, adding a catalyst, reacting for 4-10 hours, washing the product for 2-3 times respectively by suction filtration, tetrahydrofuran and ethanol, and drying at 40-80 ℃ for 6-20 hours to obtain an integrated phenylboronic acid silane reagent;
s2) modifying phenylboronic acid silane and a tail sealing reagent to the surface of spherical silica gel: starting mechanical stirring for 200-450 r/min, adding an organic solvent, adding spherical silica gel and the integrated phenylboronic acid silane reagent prepared in the step S1, starting condensed water, heating to 70-105 ℃, reacting for 4-15 hours, continuously adding a tail sealing reagent, reacting for 3-6 hours, and stopping reacting. And (3) washing the obtained product for 2-3 times by suction filtration, tetrahydrofuran and ethanol respectively, wherein the drying temperature is 40-80 ℃ and the drying time is 6-20 h, and obtaining the silica gel (marked as LBWL filler) bonded with phenylboronic acid functional groups.
Preferably, the phenylboronic acid monomer in the step S1 is 4-aminophenylboronic acid, the organosilane in the step S1 is any one of trimethoxy [2- (7-oxabicyclo [4.1.0] hept-3-yl) ethyl ] silane and triethoxy- [2- (7-oxazolicyclo [4.1.0] heptane-3-yl) ethyl ] silane, and the mass ratio of the organosilane reagent to the phenylboronic acid monomer is 4.5-10: 1. if the amount of organosilane reagent is too small, it cannot be ensured that the epoxy groups carried by the organosilane react sufficiently with the amino functions on the phenylboronic acid monomer. The excessive amount of organosilane reagent causes low reaction concentration and interferes with the reaction rate.
Further, the catalyst in the step S1 is any one or two of zinc perchlorate hexahydrate and stannous isooctanoate, preferably two of zinc perchlorate hexahydrate and stannous isooctanoate, and the mass ratio of the two is preferably 0.5: 1-3, wherein the mass ratio of the catalyst to the phenylboronic acid monomer is 1:10 to 39. The addition of the catalyst can improve the reaction efficiency of the organosilane reagent and the 4-aminophenylboric acid, improve the conversion rate of the monomer, and is preferably the mixture of the zinc perchlorate hydrate and the stannous isooctanoate, mainly because the activity of the zinc perchlorate hydrate and the stannous isooctanoate is different, and the catalyst activity and the stability of a reaction system can be considered after the zinc perchlorate hydrate and the stannous isooctanoate are mixed.
Further, the organic solvent in the step S1 is a mixed solution of ethanol and ethyl acetate or a mixed solution of tetrahydrofuran and petroleum ether, and the volume ratio of ethanol to ethyl acetate is 1:4, the volume ratio of the tetrahydrofuran to the petroleum ether is 2:3, the volume mass ratio of the organic solvent to the phenylboronic acid monomer is 5-20 mL:1g.
Preferably, in the step S2, the tail sealing agent is trimethoxysilane, and the mass ratio of the tail sealing agent to the silica gel is 1-5: 28. if the dosage of the tail sealing reagent is too small, residual silicon hydroxyl of the LBWL filler still is easy to adsorb impurities, the impurities flow out along with the target object during elution, so that the recovery rate of the target object is reduced, and trimethoxysilane cannot introduce new functional groups, so that the impurities are adsorbed.
Further, the spherical silica gel in the step S2 is ultra-pure porous spherical silica gel, the particle size range is 50-75 mu m, and the specific surface area is 350-550 m 2 /g, pore sizeThe porous spherical silica gel has higher specific surface area, and the distribution of functional groups on the surface of the silica gel is higher than that of amorphous silica gelMore uniform and has higher adsorption capacity and selectivity.
Further, in the step S2, the organic solvent is any one of dichloroethane, petroleum ether and tetrahydrofuran, and the volume mass ratio of the organic solvent to the spherical silica gel is preferably 3-20 mL:1g, wherein the mass ratio of the integral phenylboronic acid silane reagent to the spherical silica gel is 0.2-5: 1.
example 1
Mechanical stirring is started for 300 revolutions per minute, 150mL of mixed solution of ethanol and ethyl acetate (30 mL of ethanol and 120mL of ethyl acetate) is added into a 500mL three-neck flask as a solvent, 10g of 4-aminophenylboronic acid monomer and 25g of trimethoxy [2- (7-oxabicyclo [4.1.0] hept-3-yl) ethyl ] silane are added into the three-neck flask, condensed water is started and gradually heated to 90 ℃, 0.96g of catalyst zinc perchlorate hexahydrate is added for 5 hours, after reaction, the product is filtered, tetrahydrofuran and ethanol are washed for 2 times respectively, the consumption of each washing solvent is 75mL, a filter cake is dried for 12 hours through an oven, and the integral phenylboronic acid silane reagent is obtained.
The mechanical stirring is started for 300 revolutions per minute, 150mL of tetrahydrofuran is added into a 500mL three-neck flask as a solvent, then the integrated silane reagent for the first reaction and 25g of spherical silica gel are added, condensed water is started, the temperature is raised to 90 ℃, 2.5g of trimethoxysilane is added after the reaction is carried out for 6 hours, and the reaction is stopped after the continuous reaction is carried out for 4 hours. The product is filtered, washed with tetrahydrofuran and ethanol for 2 times, the dosage of the washing solvent is 75mL each time, and the filter cake is dried by a baking oven for 12 hours at 60 ℃, thus the preparation of the LBWL-1 filler is completed.
Example 2
Mechanical stirring is started for 300 revolutions per minute, 150mL of mixed solution of ethanol and ethyl acetate (30 mL of ethanol and 120mL of ethyl acetate) is added into a 500mL three-neck flask as a solvent, 10g of 4-aminophenylboric acid monomer and 50g of trimethoxy- [2- (7-oxazolbicyclo [4.1.0] heptane-3-yl) ethyl ] silane are added into the three-neck flask, condensed water is started and gradually heated to 90 ℃, 0.96g of catalyst zinc perchlorate hexahydrate is added for 5 hours, after reaction, the product is filtered, tetrahydrofuran and ethanol are washed for 2 times respectively, the consumption of each washing solvent is 75mL, a filter cake is dried for 12 hours through an oven, and the integral phenylboric acid silane reagent is obtained.
The mechanical stirring is started for 300 revolutions per minute, 150mL of tetrahydrofuran is added into a 500mL three-neck flask as a solvent, then the integrated silane reagent for the first reaction and 25g of spherical silica gel are added, condensed water is started, the temperature is raised to 90 ℃, 2.5g of trimethoxysilane is added after the reaction is carried out for 6 hours, and the reaction is stopped after the continuous reaction is carried out for 4 hours. The product is filtered, washed with tetrahydrofuran and ethanol for 2 times, the dosage of the washing solvent is 75mL each time, and the filter cake is dried by a baking oven for 12 hours at 60 ℃, thus the preparation of the LBWL-2 filler is completed.
Example 3
Mechanical stirring is started for 300 revolutions per minute, 150mL of mixed solution of ethanol and ethyl acetate (30 mL of ethanol and 120mL of ethyl acetate) is added into a 500mL three-neck flask as a solvent, 10g of 4-aminophenylboronic acid monomer and 50g of trimethoxy- [2- (7-oxazolbicyclo [4.1.0] heptane-3-yl) ethyl ] silane are added into the three-neck flask, condensed water is started and gradually heated to 90 ℃, after reaction is carried out for 5 hours, the product is filtered by suction, tetrahydrofuran and ethanol are respectively washed for 2 times, the consumption of each washing solvent is 75mL, a filter cake is dried by an oven for 12 hours, and the integral phenylboronic acid silane reagent is obtained at 60 ℃.
The mechanical stirring is started for 300 revolutions per minute, 150mL of tetrahydrofuran is added into a 500mL three-neck flask as a solvent, then the integrated silane reagent for the first reaction and 25g of spherical silica gel are added, condensed water is started, the temperature is raised to 90 ℃, 2.5g of trimethoxysilane is added after the reaction is carried out for 6 hours, and the reaction is stopped after the continuous reaction is carried out for 4 hours. The product is filtered, washed with tetrahydrofuran and ethanol for 2 times, the dosage of the washing solvent is 75mL each time, and the filter cake is dried by a baking oven for 12 hours at 60 ℃, thus the preparation of the LBWL-3 filler is completed.
Example 4
Mechanical stirring is started for 300 revolutions per minute, 150mL of mixed solution of ethanol and ethyl acetate (30 mL of ethanol and 120mL of ethyl acetate) is added into a 500mL three-neck flask as a solvent, 10g of 4-aminophenylboronic acid monomer and 50g of trimethoxy- [2- (7-oxazolbicyclo [4.1.0] heptane-3-yl) ethyl ] silane are added into the three-neck flask, condensed water is started and gradually heated to 90 ℃, 0.24g of zinc perchlorate hexahydrate and 0.72g of stannous octoate are added for reaction for 5 hours, the product is filtered by suction, tetrahydrofuran and ethanol are washed for 2 times respectively, the use amount of each washing solvent is 75mL, a filter cake is dried for 12 hours through an oven, and the integral phenylboronic acid silane reagent is obtained at 60 ℃.
The mechanical stirring is started for 300 revolutions per minute, 150mL of tetrahydrofuran is added into a 500mL three-neck flask as a solvent, then the integrated silane reagent for the first reaction and 25g of spherical silica gel are added, condensed water is started, the temperature is raised to 90 ℃, 2.5g of trimethoxysilane is added after the reaction is carried out for 6 hours, and the reaction is stopped after the continuous reaction is carried out for 4 hours. The product is filtered, washed with tetrahydrofuran and ethanol for 2 times, the dosage of the washing solvent is 75mL each time, and the filter cake is dried by a baking oven for 12 hours at 60 ℃, thus the preparation of the LBWL-4 filler is completed.
Example 5
Mechanical stirring is started for 300 revolutions per minute, 150mL of mixed solution of ethanol and ethyl acetate (30 mL of ethanol and 120mL of ethyl acetate) is added into a 500mL three-neck flask as a solvent, 10g of 4-aminophenylboronic acid monomer and 50g of trimethoxy- [2- (7-oxazolbicyclo [4.1.0] heptane-3-yl) ethyl ] silane are added into the three-neck flask, condensed water is started and gradually heated to 90 ℃, 0.24g of zinc perchlorate hexahydrate and 0.72g of stannous octoate are added for reaction for 5 hours, the product is filtered by suction, tetrahydrofuran and ethanol are washed for 2 times respectively, the use amount of each washing solvent is 75mL, a filter cake is dried for 12 hours through an oven, and the integral phenylboronic acid silane reagent is obtained at 60 ℃.
The mechanical stirring is started for 300 revolutions per minute, 150mL of tetrahydrofuran is added into a 500mL three-neck flask as a solvent, then the integrated silane reagent for the first reaction and 25g of spherical silica gel are added, condensed water is started, the temperature is raised to 90 ℃, and the reaction is stopped after the reaction is performed for 6 hours. The product is filtered, washed with tetrahydrofuran and ethanol for 2 times, the dosage of the washing solvent is 75mL each time, and the filter cake is dried by a baking oven for 12 hours at 60 ℃, thus the preparation of the LBWL-5 filler is completed.
Example 6
Mechanical stirring is started for 300 revolutions per minute, 150mL of mixed solution of ethanol and ethyl acetate (30 mL of ethanol and 120mL of ethyl acetate) is added into a 500mL three-neck flask as a solvent, 10g of 4-aminophenylboronic acid monomer and 50g of trimethoxy- [2- (7-oxazolbicyclo [4.1.0] heptane-3-yl) ethyl ] silane are added into the three-neck flask, condensed water is started and gradually heated to 90 ℃, 0.24g of zinc perchlorate hexahydrate and 0.72g of stannous octoate are added for reaction for 5 hours, the product is filtered by suction, tetrahydrofuran and ethanol are washed for 2 times respectively, the use amount of each washing solvent is 75mL, a filter cake is dried for 12 hours through an oven, and the integral phenylboronic acid silane reagent is obtained at 60 ℃.
The mechanical stirring is started for 300 revolutions per minute, 150mL of tetrahydrofuran is added into a 500mL three-neck flask as a solvent, then the integrated silane reagent for the first reaction and 20g of spherical silica gel are added, condensed water is started, the temperature is raised to 90 ℃, 2.5g of trimethoxysilane is added after the reaction is carried out for 6 hours, and the reaction is stopped after the continuous reaction is carried out for 4 hours. The product is filtered, washed with tetrahydrofuran and ethanol for 2 times, the dosage of the washing solvent is 75mL each time, and the filter cake is dried by a baking oven for 12 hours at 60 ℃, thus the preparation of the LBWL-6 filler is completed.
FIG. 1 is a scanning electron microscope image of example 1, showing that the filler is spherical and has a particle size of about 50 to 60 μm; fig. 2 is a liquid chromatogram of ribavirin standard, fig. 3 is a liquid chromatogram of example 2 filler versus ribavirin, and fig. 4 is a liquid chromatogram of a commercial PBA filler versus ribavirin, which shows that the peak shapes of ribavirin Lin Feng after passing through the column of example filler and commercial PBA filler are substantially identical to those of the standard solution. Table 1 shows N-element analysis, particle size and BET data of ribavirin Lin Zhuanyong solid phase extraction column packing.
TABLE 1 elemental analysis, particle size and BET data for ribavirin Lin Zhuanyong solid phase extraction column packing
Test example 1
Packing the solid-phase extraction column packing of ribavirin Lin Zhuanyong prepared in examples 1-6, wherein the capacity of the solid-phase extraction column is 3mL, and each small column is filled with 100mg of packing, and the specific operation steps are as follows:
activating: 1mL of 100mmol/L formic acid solution;
balance: 3mL of 0.25mol/L ammonium acetate buffer (pH 8.5);
loading: adding 6mL of a sample solution to be loaded;
leaching: 3mL of 0.25mol/L ammonium acetate buffer solution (pH 8.5) was drained;
eluting: 3mL of 100mmol/L formic acid solution, and pumping;
parallel sample: 1 blank, 3 marked.
Adding a standard sample liquid: 100uL of 0.2 ppm/methanol intermediate solution and 10uL of 1 ppm/methanol internal standard intermediate solution are accurately added into 6mL of diluted solvent, and are uniformly mixed for sample loading.
Test example 2
Carrying out liquid phase-mass spectrometry on the eluting solutions of the examples 1-6 and commercial PBA filler subjected to the solid phase extraction treatment of the test 1, wherein the standard substance is ribavirin, the on-machine detection concentration is 20ppb, and the isotope internal standard is: ribavirin (II) 13 C 5 The standard substance has the detection concentration of 10ppb on the machine. The detection instrument is a liquid chromatograph-mass spectrometer. The instrument test standard is referred to the liquid chromatography-mass spectrometry/mass spectrometry method for measuring the ribavirin Lin Canliu in the SN/T4519-2016 animal source food. Instrument reference conditions: athena UHPLC C18 (2.1. Times.50 mm,1.8 μm); mobile phase: formic acid and ammonium acetate solution; flow rate: 0.4mL/min; sample injection amount: 10. Mu.L; scanning mode: scanning positive ions; qualitative and quantitative ion: 245.1-113 (external standard); 350.1-113.1 (internal standard).
The calculation formula of the external standard recovery rate is as follows:
x: the recovery rate of the filler to the ribavirin Lin Wai target,%;
a: peak area of ribavirin in SPE labeling eluent, mAu is min;
A 0 : peak area of ribavirin in SPE blank eluent, mAu min;
b: peak area of ribavirin, mAu min, standard on-machine solution.
The result remains the decimal point one after the other.
The calculation formula of the recovery rate of the internal standard is as follows:
y: the recovery rate of the filler to the ribavirin Lin Na target,%;
c: ribavirin Lin Feng area/ribavirin Lin Nabiao peak area in SPE labeling eluent;
C 0 : ribavirin Lin Feng area/ribavirin Lin Nabiao peak area in SPE blank eluate;
d: standard on-machine solution ribavirin Lin Feng area/ribavirin Lin Nabiao peak area;
the recovery of the filler to the ribavirin Lin De external and internal standards was calculated by comparison with the peak area of the liquid-phase spectrogram of the standard, and table 2 is the average of three replicates. As can be seen from the test results in Table 2, the recovery rates of the internal standards of the examples 1-6 and the commercial PBA filler are all 92.0% -108.4%, which shows that the experimental reliability is higher; the external standard recovery rates of examples 2,4 and 6 are all higher than those of commercial PBA filler, and unexpected application effects are obtained, which indicate that the filler prepared by the method has a great application market for ribavirin detection.
TABLE 2 recovery of ribavirin from LBWL filler
While the invention has been described with reference to the preferred embodiments, it is not intended to limit the invention thereto, and it is to be understood that other modifications and improvements may be made by those skilled in the art without departing from the spirit and scope of the invention, which is therefore defined by the appended claims.
Claims (7)
1. The preparation method of the solid phase extraction column filler special for ribavirin is characterized by comprising the following steps of:
s1) preparation of an integrated phenylboronic acid silane reagent: starting mechanical stirring for 150-500 r/min, adding an organic solvent, adding phenylboronic acid monomer and organosilane into a reaction vessel for reaction, opening condensed water, heating to 70-105 ℃, adding a catalyst, reacting for 4-10 hours, washing the product for 2-3 times respectively through suction filtration, tetrahydrofuran and ethanol, and drying at 40-80 ℃ for 6-20 hours to obtain an integrated phenylboronic acid silane reagent;
s2) modifying an integrated phenylboronic acid silane reagent and a tail sealing reagent to the surface of spherical silica gel: starting mechanical stirring for 200-450 r/min, adding an organic solvent, adding spherical silica gel and the integrated phenylboronic acid silane reagent prepared in the step S1, opening condensed water, heating to 70-105 ℃, reacting for 4-15 hours, continuously adding a tail sealing reagent, reacting for 3-6 hours, and stopping; washing the obtained product for 2-3 times respectively by suction filtration, tetrahydrofuran and ethanol, wherein the drying temperature is 40-80 ℃ and the drying time is 6-20 hours, and obtaining the silica gel bonded with phenylboronic acid functional groups;
the organic solvent in the step S1 is a mixed solution of ethanol and ethyl acetate or a mixed solution of tetrahydrofuran and petroleum ether, the volume ratio of the ethanol to the ethyl acetate is 1:4, and the volume ratio of the tetrahydrofuran to the petroleum ether is 2:3, the volume mass ratio of the organic solvent to the phenylboronic acid monomer is 5-20 mL:1 g;
in the step S2, the organic solvent is any one of dichloroethane, petroleum ether and tetrahydrofuran, and the volume mass ratio of the organic solvent to the spherical silica gel is 3-20 mL:1g.
2. The method according to claim 1, wherein the phenylboronic acid monomer in the step S1 is 4-aminophenylboronic acid.
3. The preparation method according to claim 1, wherein the organosilane in the step S1 is selected from any one of trimethoxy [2- (7-oxabicyclo [4.1.0] hept-3-yl) ethyl ] silane, triethoxy- [2- (7-oxazolicyclo [4.1.0] heptan-3-yl) ethyl ] silane, and the mass ratio of the organosilane to phenylboronic acid monomer is 4.5-10: 1.
4. the preparation method according to claim 1, wherein the catalyst in the step S1 is any one or two of zinc perchlorate hexahydrate and stannous isooctanoate, and the mass ratio of the zinc perchlorate hexahydrate to the stannous isooctanoate is 0.5: 1-3, wherein the mass ratio of the catalyst to the phenylboronic acid monomer is 1: 10-39.
5. The preparation method according to claim 1, wherein the spherical silica gel in the step S2 is ultra-pure porous spherical silica gel with a particle size ranging from 50 μm to 75 μm and a specific surface area ranging from 350 μm to 550 μm 2 And/g, pore diameter of 90-500A.
6. The preparation method of claim 1, wherein the mass ratio of the integrated phenylboronic acid silane reagent to the spherical silica gel in the step S2 is 0.2-5: 1.
7. the preparation method of claim 1, wherein the tail sealing agent in the step S2 is trimethoxysilane, and the mass ratio of the tail sealing agent to the spherical silica gel is 1-5: 28.
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| CN101234337A (en) * | 2007-11-14 | 2008-08-06 | 天津大学 | Silica matrix chromatogram packing |
| CN107126943A (en) * | 2017-05-25 | 2017-09-05 | 华东理工大学 | Phenyl boric acid modified silica-gel function chromatograph packing material, preparation method and application |
| CN112717901A (en) * | 2020-11-17 | 2021-04-30 | 广州研创生物技术发展有限公司 | Modified modification of full-porous spherical silica gel and preparation method thereof |
| CN112823875A (en) * | 2019-11-20 | 2021-05-21 | 上海安谱实验科技股份有限公司 | Phenylboronic acid solid-phase extraction column filler and preparation method thereof |
| JP2022503681A (en) * | 2019-11-20 | 2022-01-12 | 上海安譜実験科技股▲ぶん▼有限公司 | Filler for phenylboronic acid solid-phase extraction column and its manufacturing method |
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| CN101234337A (en) * | 2007-11-14 | 2008-08-06 | 天津大学 | Silica matrix chromatogram packing |
| CN107126943A (en) * | 2017-05-25 | 2017-09-05 | 华东理工大学 | Phenyl boric acid modified silica-gel function chromatograph packing material, preparation method and application |
| CN112823875A (en) * | 2019-11-20 | 2021-05-21 | 上海安谱实验科技股份有限公司 | Phenylboronic acid solid-phase extraction column filler and preparation method thereof |
| JP2022503681A (en) * | 2019-11-20 | 2022-01-12 | 上海安譜実験科技股▲ぶん▼有限公司 | Filler for phenylboronic acid solid-phase extraction column and its manufacturing method |
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