免疫亲和柱在兽药残留检测应用中的研究进展

免疫亲和柱(IAC)是一种有效的兽药残留检测净化技术,可以简化样品净化过程并且提高待测物的提取效率,近几年在兽药残留检测中得到广泛应用,并表现出良好的发展前景。本文简要叙述了IAC的原理、制备过程以及在各种抗微生物类兽药检测中的应用,就IAC对目前已研制出的抗微生物药的残留检测及净化效果进行综述,并展望了IAC在未来兽药残留检测应用中的发展趋势,为研究者提供参考和研发思路。     

液相色谱–质谱联用技术在水产品兽药残留检测中的应用

液相色谱–质谱联用技术逐渐成为水产品中兽药残留检测的重要手段。从技术原理、样品处理方法的改进和新方法的应用,以及多兽药残留同时分析等方面论述了液相色谱–质谱联用技术在水产品兽药残留检测中的应用与进展。     

鸡肉中兽药残留检测技术研究进展

鸡肉中兽药残留检测是食品安全检测关注的热点,作为目前普遍发展的样品前处理技术,超临界流体萃取、加速溶剂萃取、毛细管固相微萃取及其与色谱质谱联用检测兽药残留受到广泛关注。以鸡肉为主要检测基质对这些新技术的基本原理、特点及在兽药残留分析中的应用进行了综述,同时对兽药残留分析的研究方向进行了展望。     

高通量悬浮芯片技术检测多农兽药残留

建立了一种同时可检测多农兽药残留的高通量悬浮芯片技术.以常用的7种农兽药为待测靶标物,通过优化条件,使抗原抗体基本完全反应,操作简便快捷,最多可同时检出7种农兽药残留靶标物;实验结果表明,高通量悬浮芯片检测标准回归曲线方程和相应的决定系数R2表现良好;在对含有多种农兽药残留的不同盲样的检测中,检测浓度值与实际浓度的相对偏差较小.高通量悬浮芯片技术为多农兽药残留的快速检测提供了新方法,此系统可用于多农兽药残留实际样品的初步检测,整个检测过程仅耗时1～2 h,基本上满足了多农兽药残留检测的灵敏、特异、快速和高效的需求,具有广阔的应用和发展前景.     

食品中99种兽药的一步式提取净化体系

建立了99种禁限用兽药的一步式提取净化体系,并通过高效液相色谱-四极杆/静电场轨道阱高分辨质谱（UHPLC-Q-Orbitrap HRMS）对其效果进行了评价。该提取净化体系基于载体辅助液液萃取技术,通过一次性前处理,完成常见的理化性质差异很大的8大类共计99种兽药残留的提取、净化工作,同时结合四极杆静电场轨道阱高分辨质谱实现了99种兽药残留的一步式多残留筛查。采用此体系对样品禁限用兽药进行测定分析,结果表明,该方法对液态乳、猪肉、鱼类等食品基质具有较强的适用性,检测的99种兽药线性范围为0.001~0.1 μg/mL,定量限为0.5~20.0 μg/kg,加标回收率为60%~120%,相对标准偏差小于15%。该方法的前处理和仪器分析过程对不同理化性质的化合物的兼容性强,检测效率高,可操作性强,检出限能满足所有受试的目标物,并且大大降低了检测成本。     

表面增强拉曼光谱在动物源性食品兽药残留检测的研究进展

动物性食品安全问题日益突出,愈演愈烈,有关问题食品的报道也受到了公众的广泛关注。一旦兽药在畜牧养殖过程中被过度使用或滥用,药物残留将给人类健康带来严重危害。常见的残留检测方法存在成本高、速度慢和效率低等缺点,通常需要专业人员操作,传统的理化方法已不能满足食品质量检测的需要,因此研发快速高效的兽药残留检测方法起着关键作用。近年来,表面增强拉曼光谱（SERS）技术在真伪鉴别、非法添加物检测和农兽药残留等诸多领域得到了广泛应用,并以其快速检测、无损等优势在食品检测领域有着更为广阔的发展前景。与其他检测方法不同的是,SERS通过信号放大和高灵敏度的指纹光谱,可以快速识别目标分析物,进行定性或半定量分析。本文主要介绍了拉曼光谱和SERS在动物源性食品中兽药残留检测中的研究和应用,重点介绍了SERS的优势、食品样品的前处理以及SERS结合其他技术检测多种兽药的相关研究,并提出了SERS在实际监测中面临的挑战。     

三重四极杆复合线性离子阱质谱联用技术在农兽药残留检测中的研究进展

三重四极杆复合线性离子阱质谱(QTRAP-MS/MS)联用技术可提供多种扫描模式,在保留良好选择性和灵敏度的同时提高仪器定性检测能力,在农兽药残留检测中具有独特优势。本文综合分析了近五年QTRAP-MS/MS在国内外农兽药残留检测中的应用现状,从QTRAP-MS/MS的结构及原理和检测前处理等方面展开概述,并对其未来发展趋势从前处理技术、微型便携化仪器、与其他仪器联用等方面进行展望,以期为食品安全、农药兽药残留检测提供参考。     

快速检测畜禽肉中四环素等133种兽药残留

建立了畜禽肉中133种兽药残留的快速检测方法,在避免使用缓冲盐的情况下实现了四环素与其他兽药的同时分析。样品经乙腈-水(4:1,V/V)提取,乙腈饱和正己烷和C18净化,适当氮吹浓缩后定容,再经乙腈饱和正己烷净化并高速离心,过0.22μm PTFE滤膜,LC-MS/M S分析。采用ACQUITYUPLC HSS T3色谱柱,梯度洗脱分离,电喷雾离子源(ESI),动态多重反应监测(Dynamic MRM)扫描。133种兽药在各自线性范围内,线性关系良好(r~20.99),定量限在0.05~50μg/kg之间。该方法适合于畜禽肉中多兽药残留的快速检测和定量。     

高效液相色谱-串联质谱在兽药残留分析中的应用

高效液相色谱-串联质谱以其特有的快速高效分离和定性、定量准确等优势,在兽药残留检测领域应用广泛。本文系统地介地绍了高效液相色谱-串联质谱(HPLC-MS/MS)在兽药残留分析中的研究进展,并对该领域今后的发展趋势进行了展望。引用文献66篇。     

QuEChERS-超高效液相色谱-串联质谱法测定动物源食品中4类29种禁限用兽药残留

采用改进的QuEChERS方法提取和净化鸡肉、鸡肝、猪肉、猪肝样品,建立了4类29种禁限用兽药残留的超高效液相色谱-串联质谱(UPLC-MS/MS)检测方法。样品采用McIlvaine缓冲液-乙腈提取,提取液酸化、盐析后取乙腈相,用十八烷基硅烷(C18)和氨基(NH₂)吸附剂分散固相萃取净化,最后用电喷雾串联质谱仪在正离子多反应监测扫描(MRM)模式下进行测定,基质外标法定量。解决了四环素类兽药难与其他类兽药同时检测的难题。29种兽药在各自的浓度范围内,浓度与峰面积线性关系良好,相关系数不小于0.993。在29种兽药的定量限(LOQ)水平、20 μg/kg和50 μg/kg 3个添加水平下进行加标回收率试验,29种兽药的平均回收率在71.5%和93.2%之间,相对标准偏差在0.8%和7.7%之间。29种兽药的检出限为0.3~3.0 μg/kg,金刚烷胺的定量限为1.0 μg/kg,四环素类兽药的定量限为10.0 μg/kg,其他兽药的定量限均为5.0 μg/kg。该方法操作简便,净化效果好,灵敏度、准确度和精密度均符合多残留检测技术的要求,可为食品检验机构对动物组织中禁限用兽药残留的日常监控提供更方便、快捷的检测方法支持。     

乳制品中兽药多种类残留的液相色谱-质谱分析研究进展

近年来,乳制品安全问题备受关注,兽药残留作为化学危害的一个重要部分使得针对其检测技术的研究一直是乳制品安全分析的热点领域,液相色谱-质谱技术因在灵敏度和选择性方面的优势已成为目前兽药残留分析的主流技术。基于液相色谱-质谱技术的兽药残留分析趋势已逐渐在向多种类、多组分发展,但由于兽药各种类之间在理化性质、残留状态、限量要求等方面差异较大,导致在样品前处理和仪器分析环节存在一定的技术困难。为进一步了解该技术领域的研究进展,该文从样品前处理、色谱-质谱检测和基质效应等方面对近几年采用液相色谱-质谱技术测定乳制品中兽药多种类残留的国内外文献进行了综述。     

化学发光联用技术在兽药残留分析中的应用进展

本文对近年来化学发光联用技术在兽药残留分析中的应用进展进行了评述,主要包括化学发光免疫分析、分子印迹-化学发光分析、微流控芯片-化学发光检测等,引用文献31篇.     

Validation of a confirmatory method for the determination of sulphonamides in muscle according to the European Union regulation 2002/657/EC

A simple multiresidue method is described for assaying 10 sulphonamides (SAs) (sulfadiazine, sulfathiazole, sulfapyridine, sulfamerazine, sulfamethazine, sulfamonomethoxine, sulfachlorpyridazine, sulfamethoxazole, sulfaquinoxaline and sulfadimethoxine) in muscle samples. Samples were prepared by homogenizing the tissue, extracting with ethyl acetate and cleaning up with a cation-exchange solid-phase extraction (SPE) column. The detection of analytes was achieved by HPLC-diode array detection (DAD) at 270 nm. The procedure was validated according to the European Union regulation 2002/657/EC determining specificity, decision limit, detection capability, trueness and precision. The results of validation process demonstrate that the method is suitable for application in European Union statutory veterinary drug residue surveillance programmes.     

高效液相色谱法测定动物组织样品中黄曲霉毒素的残留量

采用免疫亲合技术,对动物肝脏和肌肉中黄曲霉毒素残留进行了提取和纯化处理,建立了一种快速、灵敏、简便的测定动物组织中黄曲霉毒素B1、B2、G1、G2的高效液相色谱方法。用反相HPLC分离,柱后光化学衍生,荧光检测器测定黄曲霉毒素含量,保留时间定性,峰面积定量,测定了样品和标准样。结果表明,4种毒素可在10min内完成分离;线性范围为15～1500pg;线性回归系数大于0．9998。用本方法对动物肝脏和肌肉样品进行了加标回收实验,4种黄曲霉毒素的平均回收率为68．71％～83．42％;相对标准偏差为3．51％～7．40％;检出限均达到2．65pg。     

Immunoaffinity chromatography for the sample pretreatment of Taxus plant and cell extracts prior to analysis of taxanes by high-performance liquid chromatography

The application of immunoaffinity chromatography for the purification of Taxus plant and cell extracts prior to the HPLC analysis is described. Polyclonal antibodies raised against 10-deacetylbaccatin III (10-DAB III), paclitaxel's main precursor in plant, were characterised by enzymed-linked immunosorbent assay. Immunoglobulins from selected antisera were immobilised on CNBr-activated Sepharose 4B. The immunoaffinity column was used for the purification of plant and plant cell culture extracts prior to their analysis by HPLC. Immunoaffinity chromatography enabled the selective concentration of taxoids and enhanced sample clean-up.     

Improvement of the performance of an immunoaffinity extraction method via region-specific immobilization of IgG

Using papaverine as a model target, an immunoaffinity column of high selectivity and binding capacity was prepared by utilizing covalent linkage between the Fc portion of IgG and the surface of Sepharose 4B support. Compared with the commonly used random coupling method, the binding capacity of the region-specific immobilized antibodies was increased from 0.04 to 0.2 mol of antigens/mol of antibodies and a much larger concentration factor was thus achieved. The obtained immunoaffinity column has been successfully used in pretreatment of pericarpium papaveris samples. The method offers an improved approach to immunoaffinity extraction that should be useful for purification and concentration of other targeted compounds in highly complex mixture.     

On-line immunoaffinity capillary electrophoresis based on magnetic beads for the determination of alpha-1 acid glycoprotein isoforms profile to facilitate its use as biomarker

An immunoaffinity purification method coupled on-line to capillary electrophoresis (IACE) which allows the determination of several isoforms of intact alpha-1 acid glycoprotein (AGP) in serum samples using UV detection is developed. The immunoaffinity step is based on anti-AGP antibodies (Abs) covalently bound to magnetic beads (MBs) which are captured at the inlet end of the capillary using permanent magnets placed inside the cartridge of the CE instrument. The on-line method includes injection of the MBs with the Ab bound (MBs–Ab) and their trapping by the magnets at the entrance of the separation column, injection of serum sample and capture of AGP by the Abs, release of captured AGP, focus of desorbed protein, separation of AGP isoforms, and removal of MBs–Ab. The optimization of the different factors involved in each step allowed purification, separation and detection of AGP isoforms in a single electrophoretic analysis in about 1 h. Automation, sample and reagents consumption as well as analysis time was improved compared to off-line alternatives which use purification of AGP in an immunochromatographic column and CE separation of AGP isoforms in two independent operations. The analytical methodology developed allows the separation of 10 AGP isoforms in serum samples from a healthy donor. For a serum sample, precision (expressed as relative standard deviation) in terms of corrected area percentage was better than 0.5% for each peak accounting for more than 10% of total AGP and it was better than 4.0% in terms of relative migration time of each AGP isoform considering the whole process.     

Development of a fast and simple immunochromatographic method to purify alpha 1-acid glycoprotein from serum for analysis of its isoforms by capillary electrophoresis

Alpha 1-acid glycoprotein (AGP) is a very heterogeneous glycoprotein presenting several isoforms due to variations in its polypeptidic and glycosidic moieties. Differences in AGP isoforms between healthy and diseased individuals have been related to different pathological situations such as cancer or cardiovascular diseases, among others. Capillary electrophoresis study of the role of AGP isoforms as biomarkers requires prior purification of AGP from biological samples. Current AGP purification methods are time- and labour-consuming, and generally they have not been proven to be compatible with capillary electrophoresis analysis. In this work, different methods for AGP purification from human serum are developed and compared. The applicability of acidic precipitation and immunoaffinity chromatographic methods for AGP purification are studied. Two different immunoaffinity approaches are employed; in the first one, interferents present in the AGP sample are captured and removed, and in the second one, AGP is retained in a house-made anti-AGP column, being in this way isolated from the rest of interferents of the sample. Best results in AGP purification from human serum to be analyzed by capillary zone electrophoresis (CZE) were obtained when acidic purification was combined with immunoaffinity chromatography (IAC) employing the house-made anti-AGP column. The method was shown not to alter the proportion of AGP peaks due to isoforms existing in AGP samples. The applicability of this fast and easy purification method developed for analyzing by CZE isoforms of AGP from natural serum samples by CZE is demonstrated.     

An improved liquid chromatography/tandem mass spectrometry method for the determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA samples using immunoaffinity column purification

The analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) represents an important biomarker of oxidative stress. A sensitive method for the detection of 8-oxodG in DNA samples has been developed that utilizes immunoaffinity column purification of 8-oxodG followed by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) multiple reaction monitoring (MRM) mode analysis. An internal standard of stable-isotopically labelled 8-oxodG containing [(15)N(5)] was added prior to the enzymatic digestion of DNA to deoxynucleosides, which was then subjected to immunoaffinity column purification followed by microbore positive ion LC/MS/MS MRM. The 8-oxo-7,8-dihydroguanine (8-oxoG) base product ion at m/z 168 was monitored following cleavage of the glycosidic bond of the 8-oxodG [M+H](+) ion at m/z 284. Similar determinations were made for [(15)N(5)]8-oxodG by monitoring the [(15)N(5)]8-oxoG base product ion at m/z 173 formed from the [M+H](+) ion at m/z 289. The introduction of the immunoaffinity column purification step into the method represents a significant improvement for the accurate determination of 8-oxodG since all artefactual peaks that are observed following the direct injection of digested DNA onto the LC/MS/MS system are removed. The identity of these artefactual peaks has been confirmed to be 2'-deoxyguanosine (dG), thymidine (dT) and 2'-deoxyadenosine (dA). The presence of these artefactual peaks in MRM mode analysis can be explained as a consequence of a concentration effect due to their considerably higher relative abundance in DNA compared to 8-oxodG. The highest signal intensity was observed for the artefactual peak for dA due to the fact that the adenine base formed an adduct with methanol, which is a constituent of the mobile phase. The resulting [M+H](+) ion at m/z 284 (dA m/z 252 + CH(3)OH m/z 32) gave rise to a product ion at m/z 168 following the loss of deoxyribose in MRM mode analysis. Control calf thymus DNA was digested to deoxynucleosides and unmodfied deoxynucleosides were removed by immunoaffinity column purification; the enriched 8-oxodG was determined by LC/MS/MS MRM. The level of 8-oxodG in control calf thymus DNA was determined to be 28.8 +/- 1.2 8-oxodG per 10(6) unmodified nucleotides (n = 5) using 5 microg of digested DNA. The limit of detection of the microbore LC/MS/MS MRM for 8-oxodG was determined to be 25 fmol on-column with a signal-to-noise ratio of 3.5.