UPLC-MS/MS法快速测定水产品中硝基呋喃类代谢物残留

建立了水产品种硝基呋喃类代谢物的超高效液相色谱-串联质谱(UP-LC-MS/MS)的快速检测方法。采用2-氯苯甲醛作为衍生化试剂,使衍生化时间由以前的12 h缩短为1 h;衍生溶液用C18固相萃取柱净化,甲醇洗脱,水定容;最后采用UPLC-MS/MS检测。氨基脲(SEM)、3-氨基-2-唑烷基酮(AOZ)、5-甲基吗啉-3-氨基-2-唑烷基酮(AMOZ)、1-氨基-乙内酰脲(AHD)四种硝基呋喃类代谢物的回收率在70.1~109.3%,检出限为0.5μg/kg。     

高效液相色谱-串联质谱法快速检测海参中硝基呋喃代谢物前处理方案的探讨

利用高效液相色谱-串联质谱(HPLC-MS/MS)联合改进的QuEChERS建立了同时测定活海参中硝基呋喃类药物的代谢物氨基脲、1-氨基乙内酰脲、3-氨基-2-唑烷基酮、5-甲基吗啉-3-氨基-2-唑烷基酮的方法。样品经盐酸水解,2-硝基苯甲醛衍生,37℃水浴16 h,调节至pH 7.0~7.5,QuEChERS提取净化,氮吹至干后,用20%乙腈水定容,经C18色谱柱分离,以乙腈-0.1%甲酸溶液为流动相进行梯度洗脱,用HPLC-MS/MS以多反应监测模式进行检测。结果表明,该方法的线性范围为1.0~10μg/L,4种代谢物的相关系数均不小于0.999,检出限和定量下限分别为0.15μg/kg和0.5μg/kg,在0.5,1.0,2.0,5.0μg/kg的加标水平下,回收率为88.0%~109.6%,相对标准偏差(RSD)为3.8%~11.0%。用此方法对大连地区200批海参进行检测,合格率为95%。该方法前处理操作简便,省时,溶剂消耗量小,可作为同时分析活海参中4种硝基呋喃类药物代谢物残留量的有效手段。     

水产品中硝基呋喃代谢物残留快速检测新方法的研究

采用超高效液相色谱-串联质谱检测系统(UPLC-MS/MS)研究了2种衍生剂、2种衍生时间对水产品中4种硝基呋喃代谢物3-氨基-2-唑烷基酮(AOZ)、5-甲基吗啉-3-氨基-2-唑烷基酮(AMOZ)、氨基脲(SEM)、1-氨基-乙丙酰脲(AHD)的衍生化反应机理、衍生产物的检测灵敏度及其稳定性.质谱扫描结果表明,衍生剂2-氯苯甲醛和2-硝基苯甲醛对4种硝基呋喃代谢物的衍生化反应遵循相同的亲核加成反应机理;方差分析结果表明,同一衍生剂和同一代谢物,快速衍生化2 h和连续衍生化16 h获得的衍生产物的检测灵敏度没有显著差别;所有快速衍生物检测灵敏度连续4 d保持相对稳定.初步研究了快速水解对阳性样品的有效性.     

超高效液相色谱-串联质谱法测定猪肉中硝基呋喃类代谢物

建立了超高效液相色谱-串联质谱(UPLC-MS/MS)测定猪肉组织中4种硝基呋喃类代谢物的分析方法.样品经盐酸水解,2-硝基苯甲醛衍生,乙酸乙酯提取净化,在正离子模式下以电喷雾电离串联质谱进行测定,内标法定量.在优化的实验条件下,4种代谢物在0.5~50μg/kg范围内线性良好,相关系数大于0.995,方法检出限为0.2μg/kg,定量限为0.5μg/kg.在0.5、1.0和10.0μg/kg的添加水平下,4种代谢物的平均回收率在74.6%~104.8%之间,相对标准偏差(RSD,n=6)在2.4%~15.6%之间.方法可应用于猪肉中4种硝基呋喃类药物代谢物残留的同时检测.     

高效液相色谱-串联质谱联用测定蜂王浆中的四种硝基呋喃类药物的代谢物

报道了高效液相色谱-串联质谱联用测定蜂王浆中呋喃唑酮、呋喃西林、呋喃妥因和呋喃它酮4种硝基呋喃类药物的代谢物残留的方法。以三氯乙酸作为蜂王浆的蛋白质沉淀剂,同时提供衍生化反应所需的酸性环境;使用4种同位素内标,补偿了衍生化效率、衍生后样品溶液的pH值及光照对定量结果所产生的影响,极大地提高了定量的准确性。实验结果表明,呋喃它酮代谢物的检测下限可以达到0.03 μg/kg,其他3种硝基呋喃类药物的代谢物的检测下限可以达到0.05 μg/kg（S/N大于5）;呋喃它酮代谢物的定量下限可以达到0.20 μg/kg,其他3种硝基呋喃类药物的代谢物的定量下限可以达到0.25 μg/kg（S/N大于10）;线性范围为0.4~20 ng/mL,添加回收率为97.7%~104.8%(内标校正),相对标准偏差（RSD）为2.7%~9.7%。     

LC-MS/MS检测蜂胶中硝基呋喃类代谢物的残留

建立了蜂胶中硝基呋喃类代谢物液相色谱-串联质谱检测方法。样品经固相萃取、衍生、乙酸乙酯提取后进行质谱分析。在1.0、2.0、5.0μg/kg 3个添加水平下,硝基呋喃类代谢物的平均回收率为92.6%～99.3%,日内相对标准偏差小于10%,日间相对标准偏差小于15%。在0.5～20 ng/mL范围内呈良好的线性(r>0.99),检测限为0.25μg/kg,定量限为1.0μg/kg。方法适用于蜂胶中硝基呋喃类代谢物的分析确证。     

高效液相色谱-串联质谱法测定蜂蜡中硝基呋喃类代谢物

建立了高效液相色谱-串联质谱检测蜂蜡中呋喃唑酮代谢物(AOZ)、呋喃它酮代谢物(AMOZ)、呋喃西林代谢物(SEM)、呋喃妥因代谢物(AHD)残留的分析方法。试样采用正己烷预溶解,酸性水溶液中衍生化,经HLB固相萃取小柱净化,用Agilent Eclipse Plus-C18柱(100 mm×2.1 mm,3.5μm)分离,电喷雾离子源正离子(ESI+)、多反应监测(MRM)模式串联质谱进行测定。结果表明,4种硝基呋喃类代谢物在0.5~10 ng/m L范围内均具有较好的线性关系,相关系数大于0.995。在0.5,1.0和2.0μg/kg添加水平下,样品中4种硝基呋喃类代谢物的回收率在71.8%~119.0%之间,相对标准偏差(RSD,n=6)均小于10%,方法定量限(S/N10)为0.5μg/kg。方法适用于日常蜂蜡样品中4种硝基呋喃类代谢物残留的定性、定量分析。     

分散固相萃取-超高效液相色谱-串联质谱法测定水产品中5种硝基咪唑类和6种苯二氮卓类药物北大核心CSCD

建立了分散固相萃取-超高效液相色谱-串联质谱法同时测定水产品中5种硝基咪唑类和6种苯二氮卓类药物残留的方法。样品用1%(v/v)氨水乙腈提取,提取液经十八烷基键合硅胶(C18)和N-丙基乙二胺(PSA)吸附剂净化,在45℃下用氮气吹至近干,用1 mL甲醇-水(1∶9,v/v)溶液复溶,过0.22μm尼龙-66滤膜后用超高效液相色谱-串联质谱测定。目标化合物采用Kinetex F5色谱柱(100 mm×3.0 mm,2.6μm)分离,以0.1%(v/v)甲酸水溶液和甲醇作为流动相进行梯度洗脱,在电喷雾离子源(ESI)、正离子扫描和多反应监测(MRM)模式下进行测定,基质匹配外标法定量。结果表明,5种硝基咪唑类和6种苯二氮卓类药物在8.5 min内完成色谱分离分析,目标物在0.5~20μg/L范围内线性关系良好,相关系数均大于0.995,检出限和定量限分别为0.2~0.5μg/kg和0.5~1.0μg/kg。以草鱼、对虾和大黄鱼为样品基质,在3个不同的添加水平下,5种硝基咪唑类和6种苯二氮卓类药物的平均回收率为73.2%~110.6%,相对标准偏差(RSD)小于15%。本研究建立的方法具有简单、快速、灵敏度高和成本低等优势,可用于水产品中5种硝基咪唑类和6种苯二氮卓类药物的快速检测。该方法的建立为我国水产品质量安全相关监管部门同时监控水产品中硝基咪唑类和苯二氮卓类药物残留提供了技术支持。     

基质分散固相萃取-液相色谱-串联质谱法检测动物源性食品中硝基咪唑药物及其代谢物

建立了基质分散固相萃取-液相色谱-串联质谱（dSPE-LC-MS/MS）定量检测4种动物源性食品基质中硝基咪唑类药物及其代谢物的方法。样品（2.0 g）用乙酸乙酯提取后浓缩,经正己烷脱脂、50 mg乙二胺-N-丙基硅烷（PSA）吸附剂吸附净化后,过0.22 μm亲水聚四氟乙烯（PTFE）滤膜。采用C18柱分离,在电喷雾电离（ESI）源和选择反应监测（SRM）模式下检测,基质匹配内标法定量。在0.5~20.0 μg/L范围内,硝基咪唑类药物及其代谢物呈现良好的线性关系,相关系数（r²）>0.99;方法的检出限为0.1~0.5 μg/kg;在1.0、3.0和10.0 μg/kg的加标水平下,硝基咪唑类药物及其代谢物的回收率为84.2%~120.8%,相对标准偏差为2.0%~16.2%（n=6）。该法准确、快速,成本低,易操作,能够满足动物源性食品中硝基咪唑类药物及其代谢物残留的监测要求。     

高效液相色谱-质谱/质谱法测定蛋黄粉中硝基呋喃代谢产物

采用固相基质分散技术, 液-液分配净化, 同位素内标定量, 建立了蛋黄粉中呋喃它酮、呋喃西林、呋喃妥因和呋喃唑酮等硝基呋喃类药物代谢产物残留的高效液相色谱-质谱/质谱测定方法. 方法测定低限为0.5 μg/kg;线性范围为0.5～6.0 μg/kg;室内验证回收率范围为90.06%～109.8%;相对标准偏差2.0%～7.7%. 该方法适用于残留检测实验室对蛋黄粉类基质中硝基呋喃代谢产物的监控检测.     

Determination of total nitrofuran metabolites in shrimp muscle using liquid chromatography/tandem mass spectrometry in the atmospheric pressure chemical ionization mode

The method of MacMahon and Lohne for analysis of nitrofuran metabolites in shrimp was optimized to streamline the extraction processes and the LC analysis. This revised method includes 16 h of mild acid hydrolysis/derivatization followed by ethyl acetate extraction and analysis by LC/MS/MS in the atmospheric pressure chemical ionization mode. This revised method was validated in shrimp for concentrations of 0.25 to 2.0 ng/g. The LOQ was 0.25 ng/g for all metabolites. The LOD was 0.052 nglg for 1-aminohydantoin (AHD), 0.206 ng/g for 3-amino-2-oxazolidinone (AOZ), 0.108 ng/g for semicarbazide (SC), and 0.062 ng/g for 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ). The spike recoveries with RSD into negative matrix at 1 ng/g were 100.2% (3.2%) for AHD, 102.5% (1.0%) for AOZ, 103.7% (2.3%) for SC, and 104.0% (3.3%) for AMOZ. The spike recoveries at 1 ng/g into unknown samples (n=108) containing varied levels of nitrofuran metabolites were 112.6% (25.7%) for AHD, 108.1% (12.1%) for AOZ, 103.0% (12.0%) for SC, and 100.3% (6.9%) for AMOZ. Interday precision with samples containing incurred AOZ concentrations of 0.92 to 17.8 ppb performed over a year was 10.4% RSD. The method is accurate and precise for determining nitrofuran concentrations in the edible tissue of shrimp.     

同位素稀释质谱法测定蜂蜜中4种硝基呋喃代谢物

研究了蜂蜜中4种硝基呋喃代谢物:呋喃唑酮代谢物(AOZ)、呋喃它酮代谢物(AMOZ)、呋喃妥因代谢物(AHO)、呋喃西林代谢物(SEM)的同位素稀释HPLC-MS/MS分析方法,以邻硝基苯甲醛作为衍生化试剂,AOZ-d4、AMOZ-d5、AHD-13G3、SEM·HCl-(13C,15 N2)作内标,并将超声波衍生化法应用到实际样品的分析测试当中,以乙腈-0.1%甲酸为流动相,采用梯度洗脱,15 min可将4种代谢物完全分离,再以MS/MS进行定性和定量分析.加标回收率为82%～112%;定量限(LOQ)为0.05～1 μg/kg;检出限(LOD)为0.01～0.25 μg/kg.该方法满足欧盟(EU)对进出口蜂蜜中硝基呋喃代谢物的检测要求.     

Nitrofuran antibiotic residues in pork: The FoodBRAND retail survey

Use of nitrofuran drugs in food-producing animals has been prohibited within the EU because they may represent a public health risk. Monitoring compliance with the ban has focused on the detection of protein-bound nitrofuran metabolites which, in contrast to the parent compounds, are stable and persist in animal tissues. As part of the “FoodBRAND” project, an extensive survey of pork was undertaken across 15 European countries. Samples (n = 1500) purchased at retail outlets were analysed for the nitrofuran metabolites AOZ, AMOZ, AHD and SEM using LC–MS/MS determination of nitrobenzaldehyde derivatives. Limits of quantification for the method were 0.1 μg/kg (AOZ, AMOZ), 0.2 μg/kg (SEM) and 0.5 μg/kg (AHD). Of the 1500 samples tested, measurable residues of nitrofuran metabolites were confirmed in 12 samples (0.8% incidence overall) of which 10 samples were purchased in Portugal (AOZ, 0.3 μg/kg; AMOZ, 0.2–0.6 μg/kg) and one sample each in Italy (AMOZ, 1.0 μg/kg) and Greece (AOZ, 3.0 μg/kg).     

Development of an impedimetric immunosensor for the determination of 3-amino-2-oxazolidone residue in food samples

The use of furazolidone in food animals has been banned in European Union (EU) because of its carcinogenicity and mutagenicity on human health, but its continued misuse is widespread. Therefore, there is an urgent need for a simple, reliable, and rapid method for the detection of its marker residue, 3-amino-2-oxazolidinone (AOZ), in food products. In this regard, a sensitive and reliable electrochemical method was presented to detect AOZ based on a novel label-free electrochemical impedimetric immunosensor to address this need. The immobilization of monoclonal antibody against AOZ (denoted as AOZ-McAb) on the gold electrode was carried out through a stable acyl amino ester intermediate generated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS), which could condense antibodies on the self-assembled monolayer (SAM). The detection of AOZ was performed by measuring the relative change in charge transfer resistance before and after AOZ and AOZ-McAb immunoreaction by electrochemical impedance spectroscopy (EIS). Under the optimized conditions, the relative change in charge transfer resistance was proportional to the logarithmic value of AOZ concentrations in the range of 20.0 to 1.0 × 10⁴ ng mL⁻¹ (r = 0.9987). Moreover, the proposed immunosensor has a high selectivity to AOZ alone with no significant response to the metabolites of other nitrofuran antibiotics, such as 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), semicarbazide (SEM), and 1-aminohydantoin hydrochloride (AHD). This protocol has been applied to detect AOZ in food samples with satisfactory results.     

高效液相色谱法检测水产品中硝基呋喃类代谢物残留量

建立了水产品中硝基呋喃类代谢物残留量测定的样品处理方法和高效液相色谱分析方法.样品经酸解、2.氯苯甲醛衍生后用乙酸乙酯萃取、SPE柱净化,经高效液相-紫外检测器测定.本方法4种硝基呋喃类代谢物的定量限均为1.0μg/kg,在5.0～500μg/L质量浓度范围内,4种硝基呋喃类代谢物的工作曲线均呈良好线性,在2个添加浓度水平的平均回收率为76%～102%,相对标准偏差均小于10%(n=6).该方法适用于定性定量水产品中硝基呋喃类代谢物的残留分析.     

Nitrofuran antibiotic metabolites detected at parts per million concentrations in retina of pigs--a new matrix for enhanced monitoring of nitrofuran abuse

Nitrofuran metabolite residues AOZ, AMOZ, AHD and SEM were detected at parts per million concentrations in retina of pigs fed therapeutic doses of nitrofuran antibiotics. Discovery of this residue depot may allow widespread technology transfer to laboratories lacking LC-MS/MS thus improving global monitoring of these drugs.     

Validation of a confirmatory method for the determination of residues of four nitrofurans in egg by liquid chromatography-tandem mass spectrometry with the software InterVal

A method for the detection and determination of nitrofuran derivatives in egg by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was validated with the software InterVal and can be applied for the confirmation of nitrofuran metabolites in fresh or lyophilised eggs. The validation study comprises variations in operator, storage condition, breeding, equipment and duration of sample preparation. A comprehensive overview of the robustness of the method is obtained by analysing eight samples at six concentration levels. First results of short- and medium-term investigations for stability of analytes in solution show that standard solutions of nitrofuran metabolites are stable for at least 1 year when stored at +4 degrees C in the dark. The decision limit CCalpha expressed for the underivatised metabolite is 0.05 microg kg(-1) for 3-amino-5-methyl-morpholino-2-oxazolidinone, 0.03 microg kg(-1) for 3-amino-2-oxazolidinone, 0.20 microg kg(-1) for semicarbazide and 0.22 microg kg(-1) for 1-amino-hydantoin.     

Production and characterisation of polyclonal antibodies to a derivative of 3-amino-2-oxazolidinone, a metabolite of the nitrofuran furazolidone

Polyclonal antibodies were produced to detect 3-amino-2-oxazolidinone (AOZ), a stable metabolite of the nitrofuran antibiotic furazolidone, following derivatisation with o-nitrobenzaldehyde. A carboxyphenyl derivative of AOZ was prepared, purified and conjugated to immunogenic carrier protein. Six antisera were produced from the immunisation of seven rabbits using various immunogen doses and time-scales. IC₅₀ values, as determined by competitive enzyme-linked immunosorbent assay (ELISA) suggested that reducing immunogen dose from 0.3 to 0.05 mg, while lengthening rest periods between booster immunisations from 2 to 8 weeks, increased the sensitivity of the antibodies to 3-{[(2-nitrophenyl)methylene]amino}-2-oxazolidinone (NPAOZ) from 3.8 to 0.3 μg l⁻¹. An IC₅₀ of 0.065 μg l⁻¹ (AOZ in the form of NPAOZ) was achieved with antiserum R670 by altering ELISA conditions. This antibody was highly specific for NPAOZ and did not cross-react with various nitrofuran metabolites, their nitrophenyl derivatives or a range of veterinary drugs. Antibody R670 is suitable for incorporation into an immunoassay for AOZ with sufficient sensitivity to satisfy current criteria for monitoring of veterinary drug residues. This is the first publication of an antibody for detection of a nitrofuran metabolite.     

Determination of the furaltadone metabolite 5-methylmorpholino-3-amino-2-oxazolidinone (AMOZ) using liquid chromatography coupled to electrospray tandem mass spectrometry during the nitrofuran crisis in Portugal

The use of nitrofuran veterinary drugs as antibacterial compounds in food-producing animals has been banned in the EU since 1995. As nitrofurans are extensive and rapidly metabolized, control of their illegal use in animal production must be done in edible tissues by LC-MS/MS analysis in order to determine persistent tissue-bound metabolites. The introduction during 2002 of the multi-residue detection of nitrofuran tissue-bound metabolites by LC-MS/MS for nitrofuran control in Portuguese Residues Monitoring Plan, revealed the presence of 5-morpholinomethyl-3-amino-2-oxozolidinone (AMOZ), the bound residue of furaltadone, in a large number of samples, namely in meat poultry samples. From the 226 analysed samples in the last 4 months of 2002, 78 were non-compliant due to the presence of AMOZ (61 broilers, 11 turkeys, 5 quails and 1 pig). In this context, the aim of this paper is to describe the analytical data obtained on meat samples collected from various animal species under official Portuguese control for nitrofuran drug residues during the so-called “Portuguese nitrofuran crisis”. Presented at the AOAC Europe Workshop, November 2006, Limassol, Cyprus.     

超高效液相色谱-串联质谱法测定稻田水产品中毒死蜱残留

以QuEChERS作为样品前处理手段，采用超高效液相色谱-串联质谱（UPLC-MS/MS）检测技术，建立了稻田水产品中毒死蜱残留的检测方法。样品经乙腈提取，由0.2 g乙二胺-N-丙基硅烷（PSA）和1.2 g无水硫酸镁分散萃取净化，采用Hypersil GOLD C₁₈色谱柱（100 mm×2.1 mm，5 μm）进行分离，用加热大气压电喷雾电离源、正离子模式进行扫描，在选择反应监测模式下检测，基质匹配标准曲线外标法定量。结果表明，毒死蜱在0.5~100.0 μg/L范围内线性关系良好，相关系数大于0.999；毒死蜱的加标回收率为86.2%~103.6%，相对标准偏差为3.5%~7.6%（n=6），检出限为0.25 μg/kg，定量限为0.5 μg/kg。该方法简单、快速、灵敏，能够满足稻田水产品中毒死蜱残留的检测需求。