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taiyanhua

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注册: 2013-03-08
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专业: 高分子科学

[求助] 沉淀聚合孔雀绿染料分子印迹固相萃取-HPLC 水产品

2.3.1. Synthesis of the molecularly imprinted polymers
MG imprinted MIPs as well as non-imprinted polymer (nMIP)
was prepared by precipitation polymerization. The molar amounts
of MG as template molecule and acetonitrile as porogen used for
the preparation of MIPs and nMIP were listed in Table 1. For a general
polymerizing procedure, 0.1mmol template MG and 0.4mmol
monomer MAA were diluted in 10mL acetonitrile in a 25mL glass
tube and pre-polymerized at ambient temperature and rotated at
150rpm for 12 h. A cross-linker EDMA (2 mmol) and free-radical
initiator AIBN (20 mg) were added in the mixture solution. The
solution was degassed in an ultrasonic bath for 5 min then sparged
with oxygen-free nitrogen for 10min. The tubewas then sealed and
attached to the platform shaker and rotated slowly (about 125 rpm).
The polymerization was induced by heat irradiation at 65 ◦C for
24 h. The microspheres were extracted using a Soxhlet apparatus
in methanol–acetic acid 9:1 (v/v) for 24 h to remove the template.
Then the microspheres were washed by acetonitrile for five times
and dried in vacuum overnight at ambient temperature. nMIP was
prepared under identical conditions except that the template was
omitted.
2.3.2. Morphology observation
The surface morphology of the particles was observed by a
Hitachi S-520 field emission scanning electron microscope (Tokyo,
Japan). All samples were prepared by wetting the slide glass with a
small drop of diluted particle dispersion. Before scanning electron
microscopy (SEM) experiments, the dried specimen was coated
under vacuum with a thin layer of gold.
2.3.3. Selectivity evaluation
MIP1 was selected for subsequent experiments. The MIP1 was
packed into a 20mm×3.9mm stainless steel column by a slurry
packing technique and then coupled to the HPLC system in combination
with a post-column unit for oxidation of LMG and LGV.
The 10L solution of MG (0.251gmL−1), LMG (0.254gmL−1),
GV (0.342gmL−1), and LGV (0.251gmL−1)was injected into the
MIP1 column. 55% methanol for the first 5 min, then 80% methanol
for 3 min, and at last 100%methanol for 12minwere used as mobile
phase. The flow rate was 1.0mLmin−1.
2.3.4. Extraction mechanism
The 100mg of dry particles of MIP1 and nMIP were packed
into a 3.0mL polypropylene SPE column. The column was capped
at both ends with frits to prevent particles from leaking. Then
column was preconditioned by the following sequence: 3.0mL
methanol, 3.0mL water and 3.0mL 0.02 mol L−1 phosphate buffer
(pH 6.5). MG and its analogues were prepared in the methanoland 0.05 mol L−1 pH 4.5 ammonium acetate buffer (40:60, v/v)
with hydroxylamine hydrochloride to final concentrations of
MG (25.1gL−1), LMG (25.4gL−1), GV (34.2gL−1), and LGV
(25.1gmL−1) from the stock solutions. After conditioning, dry
MISPE columnwas loaded with 2mL above solution. Then, vacuum
was applied through the cartridges for 2 min in order to remove
residual solvent. Finally, 2mL methanol containing 0.02 mol L−1
phosphate buffer was used to elute the analytes for HPLC analysis.
2.4. Sample preparation
The grass carp, shrimp and shellfish were purchased from local
supermarket. The edible tissueswere homogenized using an Ultraturrax
(IKA-werke, Staufen, Germany) and frozen at −20 ◦C before
analysis. Homogenized blank tissue samples were spiked with
MG, LMG, GV and LGV, and left to stand for 15min at ambient
temperature prior to extraction. 5.0 g homogenate sample
were vortex-mixed with 1.5mL 20% hydroxylamine hydrochloride
and 5.0mL 0.05moLL−1 ammonium acetate buffer (adjusted to
pH 4.5 with acetic acid) in a 50mL screw-capped polypropylene
tube. Subsequently, 10mL acetonitrile were added. After vortexmixing
for 30 s, tissues were extracted for 15min on a platform
shaker (KS501 IKA, Germany) operating at 500 rpm. 10 g neutral
alumina was added into a tube and vortex-mixed for 30 s
again. The suspension was centrifuged for 5 min at 2200×g at
5 ◦C (Hitachi CR22G, Japan), and the supernatant was collected in
a 100mL round-bottom flask. Residual material was mixed with
10mLacetonitrile again. The supernatantswere combined and concentrated
on a rotor-evaporator under reduced pressure at 45 ◦C
to approximately 1 mL. Then the round-bottom was washed with
two sequential portions of 2mL of mixture containing methanol
and 0.05 mol L−1 pH 4.5 ammonium acetate buffer (40:60, v/v)
and each wash was loaded onto the 3.0mL MISPE column. The
columns were preconditioned by the following sequence: 3.0mL
methanol, 3.0mL water and 3.0mL 0.02 mol/L phosphate buffer
(pH 6.5). The columns were washed with 3.0mL water and then
with 3.0mL of a methanol:0.02 mol/L pH 6.5 phosphate buffer
(50:50, v/v), followed by a vacuum of 2min prior to elution of
the analytes with 3.0mL methanol containing 10% acetic acid.
The extracts were dried under a stream of nitrogen gas at 45 ◦C.
Finally, the residue was dissolved in 0.5mL of mixture containing
methanol and 0.05 mol L−1 pH 4.5 ammonium acetate buffer
(40:60, v/v) and centrifuged for 5 min at 2200×g before HPLC analysis.
2.5. Method validation
The evaluation of the feasibility of the multi-residue extraction
for the determination of MG, GV, and their metabolites residues
in edible muscles was carried out in accordance with the Commission
Decision 2002/657/EC [29]. Quantification was performed
using external standard calibration curve.
2.5.1. Accuracy, repeatability, within-laboratory reproducibility,
and specificity
Three sets, each of six, of blank edible muscle samples were
fortified at 1.0, 1.5 and 2.0gkg−1 of MG, GV, LMG and LGV.
The relative standard deviations (RSDs) of concentrations were
calculated for repeatability evaluation. To estimate the withinlaboratory
reproducibility, three sets, each of six, of blank samples
were fortified at 1.0, 1.5 and 2.0gkg−1 of MG, GV, LMG, and
LGV and analyzed on each of 3 days with the different instruments
and the different operators. To establish the specificity of
themethod, blank grass carp, shrimp and shellfish samples, eight of
each, were analyzed. Besides, known amounts of chloramphenicol,
3-amino-2-oxazolidinone, semicarbazide, 1-aminohydantoin and
5-morpholinemethyl-3-amino-2-oxazolidinone were added into
blank samples to evaluate possible interferences that may occur
in the method.
2.5.2. Robustness, stability, matrix calibration curve, decision
limit and detection capability
The different aquatic products were tested at ambient temperature
(the temperature varied from 15 ◦C to 20◦C) to evaluate the
robustness. The stability of MG, GV, LMG, and LGV in solution and
matrix was investigated. The stability of MG (0.251gmL−1), LMG
(0.254gmL−1), GV (0.342gmL−1), and LGV (0.251gmL−1)
in standard solution was investigated in the mixture containing
methanol and 0.05 mol L−1 pH 4.5 ammonium acetate buffer
(40:60, v/v) with hydroxylamine hydrochloride solution stored
at 4 ◦C and 20 ◦C in amber flasks. The stability of MG, GV,
LMG, and LGV in matrix was investigated in blank samples fortified
at a level of 10gkg−1 stored at −20 ◦C. The samples
were analyzed when they were fresh and after 1, 2, 3 and 4
weeks.
Furthermore, blank edible muscle samples were fortified with
working standard solutions of MG, GV, LMG, and LGV, and then
extracted to produce calibration curves with points equivalent to
1, 2, 5, 10 and 25gkg−1 of MG, GV, LMG, and LGV, respectively.
The calibration curves were obtained by plotting the recorded
peak areas versus the corresponding concentrations of the fortified
samples. The linearity of the calibration curves was expressed
by the correlation coefficient. Values of the decision limit (CC˛

and the detection capability (CCˇ) were determined by the matrix
calibration curve procedure following ISO 11843 [30]. The corresponding
concentration at the intercept plus 2.33 times the
standard deviation of the within-laboratory repeatability of the
intercept equals the decision limit. The corresponding concentration
at the decision limit plus 1.64 times the standard deviation of
the within-laboratory repeatability of the mean measured content
at the decision limit equals the detection capability. CC˛ was calculated
with a statistical certainty of 1−˛ (˛= 0.01), and CCˇ was
calculated with a statistical certainty of 1−ˇ (ˇ = 0.05) to detect
the concentration below the minimum required performance limit
(MRPL).

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zhaogenlong

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第一次看到用分子印迹做孔雀石绿的前处理，想学习一下儿

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