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taiyanhua铁虫 (初入文坛)
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沉淀聚合 孔雀绿染料分子印迹 固相萃取-HPLC 水产品
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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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