Note: Descriptions are shown in the official language in which they were submitted.
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
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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
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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.
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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.
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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.
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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
1-1111
myg cm3/g
Example
5.99 1.636 1.04 62.286 338.363 63.51 0.537
1
Example
5.87 1.631 0.99 67.011 319.27 66.29 0.529
2
Example
5.86 1.53 0.89 68.573 338.739 65.59 0.555
3
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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.
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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,
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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
PBA 86830 76320
sample
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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
