QuEChERS/UPLC-MS/MS法测定水产品中孔雀石绿和结晶紫及其代谢物

建立了水产品中孔雀石绿(MG)和结晶紫(CV)及其代谢物隐色孔雀石绿(LMG)和隐色结晶紫(LCV)残留的QuEChERS/UPLC-MS/MS分析方法。样品采用乙腈提取,改进的QuEChERS(EMR-Lipid)分散固相萃取净化,经Agilent Eclipse Plus C_(18)(1.8μm,3.0 mm×100 mm)色谱柱分离,电喷雾串联四极杆质谱多反应监测正离子方式测定。4种分析物在0.2~10.0μg/L范围内线性关系良好,相关系数均大于0.997。鱼肉中4种分析物在0.5,1.0,5.0μg/kg加标浓度水平下,回收率为77.1%~106.6%,相对标准偏差(RSD)为1.3%~4.3%。该方法简单、稳定、可靠,能有效去除样品中的蛋白质、脂肪等大分子杂质,可满足水产品中孔雀石绿、结晶紫以及隐色代谢物残留检测与确证的需要。     

液相色谱法同时测定水产品中孔雀石绿和结晶紫残留

用液相色谱-可见法同时测定水产品中孔雀石绿(MG)、结晶紫(CV)及其代谢物隐色孔雀石绿(LMG)和隐色结晶紫(LCV)的残留量,并用液相色谱-串联质谱法进行确证和定量。样品用乙腈提取,二氯甲烷液液分配,MCX阳离子固相萃取小柱净化,浓缩定容。以乙酸铵缓冲溶液和乙腈为流动相,经C18柱分离后,PbO2柱后衍生;用二极管阵列检测器在618nm测定孔雀石绿和隐色孔雀石绿,在588nm测定结晶紫和隐色结晶紫;并用串联质谱在电喷雾-多反应监测离子的模式下,进行质谱确证和定量;外标法定量,内标亮绿和氘代隐色孔雀石绿校正回收率。液相色谱-可见法的检出限为MG0.22,LMG0.28,CV0.22,LCV0.25μg/kg;液相色谱-串联质谱法的检出限为MG0.014,LMG0.018,CV0.014,LCV0.0084μg/kg。在2~20μg/kg范围内,回收率为75%~95%。     

超高效液相色谱-串联质谱法测定水产品中孔雀石绿、结晶紫及其代谢物残留量

采用超高效液相色谱-串联质谱法同时检测水产品中孔雀石绿、结晶紫及其代谢物(隐色孔雀石绿、隐色结晶紫)。经匀浆处理的水产品样品,用乙腈提取,加入酸性氧化铝去除油脂,旋转蒸发器蒸干后,用甲酸-乙腈-水(0.1+10+89.9)溶液溶解,样品溶液用超高效液相色谱分离,电喷雾串联四极杆质谱进行检测。以氘代孔雀石绿、氘代隐色孔雀石绿为内标物。孔雀石绿、结晶紫及其代谢物的质量浓度均在5.0μg·L-1以内与其峰面积呈线性关系,检出限(3S/N)在0.10～0.12μg.kg-1之间。以空白水产品样品为基体进行回收试验,方法的回收率在90.2%～108.0%之间,相对标准偏差(n=6)在2.3%～7.6%之间。     

液相色谱-串联质谱法同时测定水产品中孔雀石绿、 结晶紫以及它们的隐色代谢物残留

采用液相色谱-串联质谱法(LC-MS/MS)同时测定水产品中的孔雀石绿、结晶紫以及它们的隐色代谢物残留。匀质后的水产品样品用乙腈和乙酸铵缓冲液提取。合并提取液,用二氯甲烷反提取,经中性氧化铝柱和PRS柱固相萃取净化。采用ZORBAX SB-C18色谱柱,并以0.5 mmol/L乙酸铵-乙腈(体积比为10∶90)混合溶液为流动相,无需使用氧化铅柱在线氧化,色谱分离后直接进入串联质谱检测器检测。采用电喷雾离子源,正离子多反应监测(MRM)模式检测。方法的检测限（S/N=3）可达0.5 ng/g,平均加标回收率为77.6%～98.1%,相对标准偏差均小于8.2%。大量实际水产品样品的检测结果表明,此方法适合于对水产品中孔雀石绿、结晶紫以及它们的隐色代谢物的残留检测。     

高效液相色谱-四极杆串联线性离子阱质谱法测定水产品及饲料中的孔雀石绿、结晶紫及其代谢物

提出了高效液相色谱-四极杆串联线性离子阱质谱法测定水产及饲料中的孔雀石绿、结晶紫及其代谢物(隐色孔雀石绿、隐色结晶紫)的方法。样品经乙腈提取,Agilent XDB C18色谱柱分离,四极杆串联线性离子阱质谱进行检测。以氘代孔雀石绿、氘代隐色孔雀石绿、氘代结晶紫、氘代隐色结晶紫为内标物。4种化合物的线性范围均为0.05~5.0μg·L-1,检出限(3S/N)均为0.5μg·kg-1。以空白样品为基体做加标回收试验,计算得4种化合物的回收率在93.3%~120%之间,相对标准偏差(n=6)在1.4%~18%之间。另外,还采用数据相关采集模式结合增强离子扫描模式对4种化合物进行定性分析,可对试样中4种化合物在定量同时进行定性确证。     

水产品中孔雀石绿、结晶紫及其代谢产物检测方法的探讨

采用低温冷冻、超高速离心技术提取水产品中的孔雀石绿、结晶紫及其代谢产物,并通过分析几种国标方法的优缺点,对方法的若干步骤进行探讨,改进了水产品中孔雀石绿、结晶紫及其代谢产物的检测方法.方法的回收率为78.3%～100.9%,检出限为0.5μg/kg,测定结果的相对标准偏差为1.3%～7.1%(n=6).该方法操作简便,...     

水产品中孔雀石绿、结晶紫及其代谢产物残留量的检测

孔雀石绿（MG）和结晶紫（CV）具有高毒素、高残留和致癌、致畸、致突变等特点,当其进人生物体内,就会产生具有更强危害的隐性孔雀石绿（LMG）和隐性结晶紫（LCV）。鉴于孔雀石绿和结晶紫的危害性,包括我国在内的许多国家都将它们列为水产养殖中的禁用药物。     

超高效液相色谱-线性离子阱/静电场轨道阱质谱快速筛查和确证化妆品中孔雀石绿、结晶紫及其代谢物

采用超高效液相色谱-线性离子阱/静电场轨道阱组合式高分辨质谱联用技术,建立了快速筛查、定性识别化妆品中的孔雀石绿、隐色孔雀石绿、结晶紫和隐色结晶紫的方法。不同剂型的化妆品样品经甲醇提取后,通过静电场轨道阱高分辨质谱全扫描得到目标化合物准分子离子的精确质量数,据此对化妆品进行快速筛查,并用离子阱的二级质谱分析对化合物进行了进一步确认,4种化合物检出限≤5μg/kg。方法适用于化妆品中孔雀石绿、隐色孔雀石绿、结晶紫和隐色结晶紫的快速筛查和确证。     

反相高效液相色谱法测定水产品中孔雀石绿、结晶紫及其代谢物

利用反相高效液相色谱研究了水产品中孔雀石绿、结晶紫及其代谢物隐性孔雀石绿、隐性结晶紫的同时测定。采用Krom asil C18色谱柱,PbO2-硅藻土柱为柱后氧化柱,以乙腈-乙酸铵缓冲溶液-冰乙酸(体积比为58∶14∶28)体系为流动相。孔雀石绿、隐性孔雀石绿、结晶紫、隐性结晶紫的加标回收率分别为84.6%、85.8%、89.8%、88.5%,相对标准偏差分别为5.0%、4.7%、4.3%、4.6%(n=6),检出限为2μg/kg。     

高效液相色谱快速测定水产品中孔雀石绿、结晶紫及其无色产物的残留量

采用高效液相色谱同时检测水产品中孔雀石绿、结晶紫及无色孔雀石绿和无色结晶紫的残留量,样品经提取、净化处理后所得残渣用乙腈溶解后,通过采用C_(18)色谱柱,以乙腈(A)和pH3.0的0.02 mol·L~(-1)磷酸二氢钾缓冲溶液(B)按不同比例混合进行梯度淋洗,实现孔雀石绿、结晶紫及其代谢物的分离。用自制的二氧化铅柱氧化无色孔雀石绿及无色结晶紫。在588 nm波长处,测定4种物质的质量浓度在0.3～6.0 mg·L~(-1)范围内与其峰面积呈线性关系,相对标准偏差(n=6)小于2.5%,检出限(3S/N)小于1.9μg·kg~(-1),分析时间20 min。以凤尾鱼罐头为基体进行回收试验,方法的回收率在71.5%～88.6%范围。     

Simultaneous determination of malachite green, brilliant green and crystal violet in grass carp tissues by a broad-specificity indirect competitive enzyme-linked immunosorbent assay

An immunizing hapten (4-(carboxymethoxy)phenyl)bis(4-(diethylamino)phenyl)methylium for brilliant green (BG), a triphenylmethane dye with a potential illegal use in fish feeding, was synthesized and used to produce polyclonal antibody (PcAb) against BG. Unexpectedly, the obtained PcAb showed high cross-reactivity (CR) to malachite green (MG) and crystal violet (CV) in an indirect competitive enzyme-linked immunosorbent assay (icELISA). After screening against three heterologous coating antigens, the icELISA exhibited good sensitivity and uniform response to BG (IC(50) of 1.98 ng mL(-1) and CR of 100%), MG (IC(50) of 1.61 ng mL(-1) and CR of 105%) and CV (IC(50) of 1.34 ng mL(-1) and CR of 142%) when using (4-(carboxymethoxy)phenyl)bis(4-(dimethylamino)phenyl)methylium as the coating hapten. Therefore, a broad-specificity icELISA for simultaneous determination of BG, MG and CV was developed. The recoveries of single analyte and mixture of three analytes from spiked grass carp tissues were estimated ranging from 74.94% to 110.39%. A statistically significant correlation of results was obtained between the developed icELISA and previously established HPLC approaches with the food-relevant three triphenylmethane dyes concentration range 1.83-200 ng mL(-1) (R(2)=0.9224), indicating good accuracy of the icELISA and suitability for the broad-specific detection of the three triphenylmethane dyes in grass carp tissues.     

Application of ionic liquid‐based microwave‐assisted extraction of malachite green and crystal violet from water samples

A simple, environment friendly and efficient technique, ionic liquid‐based microwave‐assisted extraction was first used to determine malachite green and crystal violet (CV) from water samples coupled to HPLC. The key parameters influencing extraction efficiency were investigated, such as the type of ionic liquids, the volume of ionic liquid, extraction time, and so on. Under the optimum conditions, good reproducibility of the extraction performance was obtained (RSD, 1.0% for malachite green (MG) and 5.9% for CV, n = 5). Good linearity (0.10–25 μg L^?1) was observed with correlation coefficients between 0.9991 and 0.9964. The detection limits of MG and CV were 0.080 and 0.030 μg L^?1, respectively. The proposed method had been successfully applied to determine MG and CV in real water samples with recoveries ranging from 95.4 to 102.8%. Compared with the previous technologies, the proposed method required less extraction time (2 min), and provided lower detection limits and higher enrichment factors. Moreover, there were no volatile and hazardous organic solvents released. Based on these simple, environment friendly, rapid, and highly efficient results, the proposed approach provides a new and promising alternative for simultaneously extracting trace amounts of MG and CV from water.     

Monoclonal antibody-based enzyme-linked immunosorbent assay for detection of total malachite green and crystal violet residues in fishery products

A rapid easy-to-use trace level direct competitive enzyme-linked immunosorbent assay (dc-ELISA) detection of total residual malachite green (MG), crystal violet (CV) and their corresponding primary metabolites leucomalachite green (LMG) and leucocrystal violet (LCV) in fishery products in a single assay was developed. The monoclonal antibodies, anti-MG and anti-CV mAbs, were prepared using carboxyl-malachite green (CMG) and cationized bovine serum albumin (cBSA) conjugates as immunogen. The linear range for the quantitative detection of total MG, CV and their primary metabolites LMG and LCV was between 0.15 to 4.5?ng?mL^?1 with a half maximal inhibitory concentration (IC₅₀) at 0.56?±?0.04?ng?mL^?1 (n?=?5). The anti-MG mAbs exhibited 98% cross-reactivity to CV, less than 0.1% cross-reactivity with LMG and LCV, and no cross-reactivity with chloramphenicol, enrofloxacin, sulfadiazine, and tetracycline. Application of the dc-ELISA in fish tissue samples gave a limit of detection (LOD) of 0.37?ng?g^?1. The improved total detection lead to a recovery of 74.60?±?8.38% at 0.5?ng?g^?1 and 87.47?±?12.83% at 2.0?ng?g^?1 that was better than existing techniques. The dc-ELISA showed total MG in 7 out of 44 field fish samples that were confirmed with LC-MS/MS. The easy-to-use, inexpensive, and rapid dc-ELISA for the detection of total MG, CV and their corresponding primary metabolites holds promise for field applications.     

Determination of multi-residue for malachite green,gentian violet and their metabolites in aquatic products by high-performance liquid chromatography coupled with molecularly imprinted solid-phase extraction

A sorbent was synthesized and investigated for molecularly imprinted solid-phase extraction (MISPE). Molecularly imprinted polymers (MIPs) were synthesized via precipitation polymerization procedure, where methacrylic acid (MAA) was used as functional monomer and ethylene glycol dimethacrylate (EDMA) as cross-linking agent. The imprinting effect and selectivity of the MISPE were evaluated by elution experiments. The resulting MISPE showed high extraction selectivity to malachite green, gentian violet and their metabolites, which may be caused by both the ion exchange and the hydrophobic interactions. The determination of multi-residue for malachite green, gentian violet and their metabolites in aquatic products by HPLC coupled with MISPE was also investigated. The mean recoveries calculated by solvent calibration curve for malachite green (MG), gentian violet (GV), leucomalachite green (LMG) and leucogentian violet (LGV) were from 89.8% to 99.1% for grass carp, 90.6% to 101.2% for shrimp and 91.3% to 96.3% for shellfish. The decision limit (CCα) and the detection capability (CCβ) obtained for MG, GV, LMG and LGV were in the range of 0.11–0.14 and 0.19–0.24 μg kg⁻¹ for grass carp, shrimp and shellfish. The MISPE was successfully used off-line for the determination of MG, GV and their metabolites in aquatic products.     

水滑石改性二氧化硅新材料去除养殖用水中的孔雀石绿及结晶紫

合成了水滑石改性的系列硅胶新材料HTS-n(s),该新材料同时包含有酸性位与碱性位,并通过碱腐蚀获得了3～4 nm的小孔和大的比表面积。该类材料可选择性吸附去除水产养殖用水中的工业染料类污染物孔雀石绿和结晶紫。吸附后水中剩余的染料浓度通过高效液相色谱-质谱/质谱联用仪进行检测,结果显示新材料的吸附性能超过常用的工业吸附剂NaY沸石和活性炭。     

Determination of malachite green residues in fish using molecularly imprinted solid-phase extraction followed by liquid chromatography-linear ion trap mass spectrometry

This paper describes the development of an analytical procedure to determine malachite green (MG) residues in salmon samples using molecularly imprinted polymers (MIPs) as the extraction and clean-up material, followed by liquid chromatography-linear ion trap mass spectrometry (LC-QqQLIT-MS/MS). MG and two structurally related compounds, crystal violet (CV) and brilliant green (BG) were employed for the selectivity test. The imprinted polymers exhibited high binding affinity for MG, while CV and BG showed less binding capacity: 47% and 34%, respectively. The recovery values of MG in salmon samples fortified with leucomalachite green (LMG) were determined by measuring the amount of MG in the sample, after carrying out the oxidation reaction with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), which converts the LMG back into chromic-form. The average recovery of MG in spiked salmon muscle over the concentration range 1-100 ng g⁻¹ was 98% with a relative standard deviation value (R.S.D.) below 12%. The method detection limits (MDLs) obtained for MG, CV, BG and their leuco-metabolites were in the range of 3-20 ng kg⁻¹ (ppt).     

Confirmatory analysis of malachite green, leucomalachite green, crystal violet and leucocrystal violet in salmon by liquid chromatography-tandem mass spectrometry

A method has been developed to analyse for malachite green (MG), leucomalachite green (LMG), crystal violet (CV) and leucocrystal violet (LCV) residues in salmon. Salmon samples were extracted with acetonitrile:McIIIvain pH 3 buffer (90:10 v/v), sample extracts were purified on a Bakerbond strong cation exchange solid phase extraction cartridge. Aliquots of the extracts were analysed by LC-MS/MS. The method was validated in salmon, according to the criteria defined in Commission Decision 2002/657/EC. The decision limit (CCalpha) was 0.17, 0.15, 0.35 and 0.17 microg kg(-1), respectively, for MG, LMG, CV and LCV and for the detection capability (CCbeta) values of 0.30, 0.35, 0.80 and 0.32 microg kg(-1), respectively, were obtained. Fortifying salmon samples (n=6) in three separate assays, show the accuracy to be between 77 and 113% for MG, LMG, LCV and CV. The precision of the method, expressed as RSD values for the within-laboratory reproducibility, for MG, LMG and LCV at the three levels of fortification (1, 1.5 and 2.0 microg kg(-1)), was less than 13%. For CV a more variable precision was obtained, with RSD values ranging between 20 and 25%.     

Fabrication of the Monolithic Fiber for the Solid Phase Micro‐extraction of Malachite Green

A monolithic fiber of molecularly imprinted polymer (MIP) was prepared by in situ polymerization within the capillary with an inner diameter of 530 µm. It was carried out in 8 min by microwave irradiation using malachite green (MG) as a template molecule, α‐methacrylic acid (MAA) as a functional monomer, acetonitrile (ACN) as a porogenic solvent, ethylene dimethacrylate (EDMA) as a crosslinker, azodiiso‐butyronitrile (AIBN) as a thermal initiator. The resulted MIP fibers were pushed out from the capillary, eluted and inserted in the capillary again, which successfully used for the solid phase microextraction (SPME) procedure. The factors affecting the extraction of MG, such as the molar ratio of template/monomer (MG/MAA), concentration of NaCl, extraction and desorption time, and extraction and desorption solvents were investigated in detail. The selectivity of the MIP fibers was compared using MG analogues crystal violet (CV) and non‐analogue Sudan II. It was also employed for the pretreatment of trace MG in the fish feed followed by high‐performance liquid chromatography (HPLC) detection. Under the optimal conditions, the linear range of MG was 10‐600 μg/L, the detection limit (LOD) was 1.23 μg/L and the recovery of spiked fish feed sample was 88.7～113.9%.     

Magnetic solid‐phase extraction for determination of the total malachite green,gentian violet and leucomalachite green,leucogentian violet in aquaculture water by high‐performance liquid chromatography with fluorescence detection

In this study, magnetic multi‐walled carbon nanotube nanoparticles were synthesized and used as the adsorbent for the sums of malachite green, gentian violet and leucomalachite green, leucogentian violet in aquaculture water samples followed by high performance liquid chromatography with fluorescence detection. This method was based on in situ reduction of chromic malachite green, gentian violet to colorless leucomalachite green, leucogentian violet with potassium borohydride, respectively. The obtained adsorbent combines the advantages of carbon nanotubes and Fe₃O₄ nanoparticles in one material for separation and preconcentration of the reductive dyes in aqueous media. The structure and properties of the prepared nanoparticles were characterized by transmission and scanning electron microscopy, X‐ray diffraction, and Fourier‐transform infrared spectroscopy. The main parameters affecting the adsorption recoveries were investigated and optimized, including reducing agent concentration, type and amount of sorbent, sample pH, and eluting conditions. Under the optimum conditions, the limits of detection in this method were 0.22 and 0.09 ng/mL for malachite green and gentian violet, respectively. Product recoveries ranged from 87.0 to 92.8% with relative standard deviations from 4.6 to 5.9%. The results indicate that the sorbent is a suitable material for the removal and concentration of triphenylmethane dyes from polluted environmental samples.