液相色谱-高分辨质谱技术在兽药残留检测中的研究进展

随着社会经济的快速发展,人民生活水平不断提高,对动物源性食品的需求量也不断增加,动物源性食品中兽药残留问题也越来越受重视,对兽药残留的监督检验工作也提出了更高的要求。高分辨质谱具有极强的化合物检索能力,能够在无标准品的情况下实现多种兽药的非靶向筛查确证,很好地弥补了三重四极杆质谱检测宽泛性差、未知化合物筛查能力欠缺的缺点,是兽药残留风险监测和高通量筛查的重要技术手段。本文通过综述2018—2021年液相色谱-高分辨质谱技术在兽药残留检测领域中的应用情况,对高分辨质谱的优势和特点进行系统阐述。随着高分辨质谱仪的普及和检测技术的发展,高分辨质谱技术将可能作为常规检测方法被广泛应用于兽药残留检测领域。     

Evaluation of a rapid screening method for chemical contaminants of concern in four food-related matrices using QuEChERS extraction, UHPLC and high resolution mass spectrometry

A method combining QuEChERS extraction, ultra-high pressure liquid chromatography and full scan high resolution mass spectrometry was evaluated for its use in screening for chemical residues and contaminants in animal-related food matrices. The method was evaluated by analysis of multiple replicates of whole milk, muscle tissue, liver tissue and corn silage. Analytes tested included plant alkaloids, carbamate and organophosphate pesticides, and several types of veterinary drugs. A database containing the chemical formula for each analyte was used to calculate accurate mass-to-charge ratios for expected pseudo-molecular ions. This information, as well as retention times, was used to identify analytes. Of 118 compounds chosen for analysis, 86 were detectable in all fortified replicates of at least one matrix at levels ranging from 1.0 to 5000?ng/g. Variability of response, as measured in % relative standard deviation of peak areas over seven replicate fortified sample analyses, was found to differ among the classes of analytes, ranging from <10% to >100%. Retention times were stable and analytes were routinely detected at measured mass-to-charge ratios within 2?ppm of their theoretical mass-to-charge ratios. These results indicate that the combination of generic extraction and chromatographic procedures with full scan high resolution mass spectrometry can serve as a useful method for screening complex matrices.     

Analytical method for fast screening and confirmation of multi-class veterinary drug residues in fish and shrimp by LC-MS/MS

A multi-class, multi-residue analytical method based on LC-MS/MS detection was developed for the screening and confirmation of 28 veterinary drug and metabolite residues in flatfish, shrimp and eel. The chosen veterinary drugs are prohibited or unauthorised compounds in Korea, which were categorised into various chemical classes including nitroimidazoles, benzimidazoles, sulfones, quinolones, macrolides, phenothiazines, pyrethroids and others. To achieve fast and simultaneous extraction of various analytes, a simple and generic liquid extraction procedure using EDTA-ammonium acetate buffer and acetonitrile, without further clean-up steps, was applied to sample preparation. The final extracts were analysed by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). The method was validated for each compound in each matrix at three different concentrations (5, 10 and 20 ng g^–1) in accordance with Codex guidelines (CAC/GL 71-2009). For most compounds, the recoveries were in the range of 60–110%, and precision, expressed as the relative standard deviation (RSD), was in the range of 5–15%. The detection capabilities (CCβs) were below or equal to 5 ng g^–1, which indicates that the developed method is sufficient to detect illegal fishery products containing the target compounds above the residue limit (10 ng g^–1) of the new regulatory system (Positive List System – PLS).     

Development and validation of a methodology to qualitatively screening veterinary drugs in porcine muscle via an innovative extraction/clean-up procedure and LC-MS/MS analysis

A qualitative multiresidue method that facilitates rapid monitoring of veterinary drugs in porcine muscle is described. The method comprises the application of an innovative extraction/clean-up procedure, namely liquid–liquid extraction with partition at very low temperature (LLE-FPVLT), and analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Besides the high selectivity, sensitivity and specificity, this high-throughput method proved to be quite general as 34 veterinary drugs (from six distinct classes: tetracyclines, sulfonamides, penicillins, quinolones, macrolides and benzimidazoles) could be successfully detected. The whole screening procedure was validated according to the directives from European Commission Decision 2002/657/EC and guidelines for the validation of screening methods. Acceptable values for the evaluation parameters were achieved for all analytes (except for ampicillin, clindamycin and erythromycin). Finally, these very promising results have strengthened the possibility of inclusion of such a methodology as an integral part of the National Residue Control Plan scope of the Ministry of Agriculture, Livestock and Food Supply of Brazil.     

Development and validation of a methodology to qualitatively screening veterinary drugs in porcine muscle via an innovative extraction/clean-up procedure and LC-MS/MS analysis

A qualitative multiresidue method that facilitates rapid monitoring of veterinary drugs in porcine muscle is described. The method comprises the application of an innovative extraction/clean-up procedure, namely liquid-liquid extraction with partition at very low temperature (LLE-FPVLT), and analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Besides the high selectivity, sensitivity and specificity, this high-throughput method proved to be quite general as 34 veterinary drugs (from six distinct classes: tetracyclines, sulfonamides, penicillins, quinolones, macrolides and benzimidazoles) could be successfully detected. The whole screening procedure was validated according to the directives from European Commission Decision 2002/657/EC and guidelines for the validation of screening methods. Acceptable values for the evaluation parameters were achieved for all analytes (except for ampicillin, clindamycin and erythromycin). Finally, these very promising results have strengthened the possibility of inclusion of such a methodology as an integral part of the National Residue Control Plan scope of the Ministry of Agriculture, Livestock and Food Supply of Brazil.     

Multiresidue method for pesticides and veterinary drugs in bovine milk using GC/MS and LC/MS/MS

A simple, sensitive and selective method with gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed to detect 342 pesticides and veterinary drugs contaminating bovine milk at the maximum residue limits (MRLs) defined in the "positive list system". Sample preparation was performed by extracting the analytes with acetonitrile, followed by salting-out with sodium chloride. For some pesticides, the extract was further cleaned up by n-hexane partitioning and PSA cartridge column chromatography. GC/MS-EI or -NCI was used to determine pesticide residues, while LC/MS/MS-ESI was applicable to the determination of pesticide and veterinary drug residues. The variation of the recoveries of these drugs at MRL was relatively wide; however the relative standard deviations of the recovery of each drug were within 28%, suggesting that the present method is good enough for use as a screening test for contaminants at the MRLs. These results show that this method is useful for multiresidue analysis of numerous pesticides and veterinary drugs in bovine milk.     

高分辨质谱定性筛查牛奶和奶粉中兽药残留

目的 建立高分辨质谱定性筛查牛奶和奶粉中兽药残留的高通量方法。方法 样品经乙腈、QuEChERS盐包提取, 采取改良的3种不同方式[QuChERS、高分辨质谱(high resolution mass spectrometry, EMR)、固相萃取(solid phase extraction, SPE)]净化提取液, 用液相色谱联用高分辨质谱(liquid chromatography-high resolution mass spectrometry, LC-HRMS)筛查了牛奶和奶粉中138种兽药残留, 建立兽药信息数据库, 并用截断值(Vcut-off, 判断阳性和阴性样品的临界值)作为评估筛查方法的指标。结果 改良的QuChERS可以获得更高的回收率, 当截断值(Vcut-off)设定为0.2时, 在2种加标水平下, 有109种兽药通过了筛查方法验证的要求, 假阳性和假阴性概率均低于5%。结论 此方法适用于牛奶和奶粉中多兽药残留的定性筛查, 一针进样可以得到109种兽药残留的筛查信息, 方法假阳性和假阴性概率在规定的目标物浓度下都低于5%。     

多种样品前处理方法在高分辨质谱法检测农药残留中的应用

食品、药品中的农药残留安全问题引起社会越来越多的关注, 农药残留检测方法为食品、药品安全监管提供技术支撑。高分辨质谱法由于具有较高的分辨率和质量精确度, 近年来在农药残留检测中的应用越来越多, 尽管高分辨质谱法能在一定程度上降低基质干扰, 但样品前处理仍然是必要的。样品前处理可以减少基质干扰成分, 提取、富集目标检测物, 对农药残留检测方法的准确度、灵敏度和重现性具有重要影响。本文对近年来高分辨质谱法检测农药残留所应用的前处理方法进行概述, 主要包括QuEChERS法、固相萃取法、极性农药快速提取法、在线前处理法等, 介绍了各前处理方法的原理和应用现状, 对不同前处理方法的优缺点进行分析, 并对高分辨质谱法检测农药残留所应用的样品前处理的发展趋势进行展望。     

Comprehensive screening for multi-class veterinary drug residues and other contaminants in muscle using column-switching UPLC-MS/MS

A quantitative multi-class analytical method covering more than 226 veterinary drugs and other contaminants in muscle, belonging to different drug families, was developed. The method is based on liquid–liquid extraction, purification by low-temperature clean-up and dispersive solid-phase extraction (D-SPE), and analysis was conducted in two analytical runs by column-switching UPLC coupled with electrospray ionisation and tandem mass spectrometry (UPLC-ESI-MS/MS). For most of the target analytes, the optimised pre-treatment processes led to no significant interference from the sample matrix. The limit of quantification varied from 0.05 to 10 µg kg^–1. Statistical evaluation indicated that average recoveries spiked into pork were in the range from 62.4% to 138.8%, and the relative standard deviations were in the range from 2.8% to 26.6% (intra-day precision). The availability of this method will contribute to a better safety assurance of meat with a significant reduction of both effort and time.     

Multiclass and multi-residue screening of mycotoxins,pharmacologically active substances,and pesticides in infant milk formulas through ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry analysis

Infant milk formulas are designed to substitute human milk when breastfeeding is unavailable. In addition to human milk and milk-derived products, these formulas can be a vehicle of contaminants. In this work, a multiclass method based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach was developed for the simultaneous determination of contaminants (n = 45), including mycotoxins and veterinary drug residues, occurring in infant milk formulas. By using an ultra-high-performance liquid chromatography quadrupole-Orbitrap coupled with high-resolution mass spectrometry analysis (UHPLC-Q-Orbitrap HRMS; Thermo Fisher Scientific), further retrospective analysis of 337 contaminants, including pesticides, was achieved. The method was validated in accordance with European regulations and applied for the analysis of 54 infant milk samples. Risk assessment was also performed. Dexamethasone was detected in 16.6% of samples (range: 0.905–1.131 ng/mL), and procaine benzyl penicillin in 1 sample at a concentration of 0.295 ng/mL. Zearalenone was found in 55.5% of samples (range: 0.133–0.638 ng/mL) and α-zearalenol in 16.6% of samples (range: 1.534–10.408 ng/mL). Up to 49 pesticides, 11 veterinary drug residues, and 5 mycotoxins were tentatively identified via retrospective analysis based on the mass spectral library. These findings highlight the necessity of careful evaluation of contaminants in infant formulas, considering that they are intended for a vulnerable part of the population.     

Analysis of veterinary drug and pesticide residues in animal feed by high-resolution mass spectrometry: comparison between time-of-flight and Orbitrap

The use of medium–high-resolution mass spectrometers (M–HRMS) provides many advantages in multi-residue analysis. A comparison between two mass spectrometers, medium-resolution (MRMS) time-of-flight (TOF) and high-resolution (HRMS) Orbitrap, has been carried out for the analysis of toxic compounds in animal feed. More than 300 compounds belonging to several classes of veterinary drugs (VDs) and pesticides have been determined in different animal feed samples using a generic extraction method. The use of a clean-up procedure has been evaluated in both instruments, and several validation parameters have been established, such as the matrix effect, linearity, recovery and sensitivity. Finally, both instruments have been used during the analysis of 18 different feed samples (including chicken, hen, rabbit and horse). Some VDs (sulfadiazine, trimethoprim, robenidine and monensin sodium) and one pesticide (chlorpyrifos) have been identified. In general, better results were obtained using the Orbitrap, such as sensitivity (1–12.5 µg kg^?1) and recovery values (60–125%). Moreover, this analyser had several software tools, which reduced the time for data processing and were easy to use, performing quick screening for more than 450 compounds in less than 5 min. However, some disadvantages such as the high cost and a decrease in the number of detected compounds at low concentrations must be taken into account.     

基于金属有机框架的农产品中农兽药残吸附萃取技术研究进展

农产品中农兽药物的残留是食品安全领域的主要问题之一,有必要对这些药物残留进行含量检测并严格限量。目前的吸附萃取材料如沸石、多壁碳纳米管等存在吸附量少、孔隙易堵塞的问题。金属有机框架（Metal-organic Frameworks,MOFs）因具有可调节孔隙、分级结构、巨大的孔体积和表面积、优异的吸附性能和可回收等特点而被逐渐应用于吸附萃取农产品中的农兽药残。本文综述了MOFs作为吸附剂吸附萃取农产品中农兽药残的应用现状,并分别从农、兽药种类的角度出发就MOFs的吸附应用进行了系统阐述,综合分析了MOFs的优异吸附性能。文章为MOFs吸附萃取农兽药残技术的进一步发展提供参考,为食品安全检测提供了技术借鉴。     

Multiclass method for fast determination of veterinary drug residues in baby food by ultra-high-performance liquid chromatography–tandem mass spectrometry

A simple and rapid multiresidue method for the determination of different veterinary drug residues in meat-based baby food (MBF) and powdered milk-based infant formulae (PMIF) has been developed. The method involves an extraction procedure based on buffered QuEChERS (quick, easy, cheap, effective, rugged and safe) methodology, without any further clean-up step, followed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The method has been validated in two baby food matrices (MBF and PMIF) at three different concentration levels, obtaining suitable recoveries and precision (inter and intra-day precision) values. Quantification was carried out using matrix-matched standard calibration. Furthermore, the decision limit (CCα) and the decision capability (CCβ) were evaluated, ranging from 0.5 to 16.2 μg/kg and from 1.2 to 22.4 μg/kg, respectively. Finally, the method was applied to the analysis of several kinds of baby food samples and traces of some veterinary drugs were detected.     

飞行时间质谱技术及其在食品安全检测中的应用

飞行时间质谱技术(TOF—MS)作为质谱技术的一种,因具有高灵敏度及高分辨率等优点,已作为高端检测手段在食品质量安全领域广泛应用。文章综述飞行时间质谱技术的发展现状,及其在食品添加剂、食品污染物、违法添加的非食用物质、农药残留、兽药残留及真菌毒素6个方面应用的国内外研究进展,并展望该技术的应用前景。     

PRiME HLB固相萃取-高效液相色谱-串联质谱法同时快速测定鸡蛋中48种兽药残留

建立固相萃取-高效液相色谱-串联质谱同时快速测定鸡蛋中48种兽药残留的分析方法。样品采用90%乙腈水溶液提取,用PRiME HLB小柱净化浓缩后用电喷雾离子源,正负离子扫描,多反应监测模式的高效液相色谱-串联质谱法进行检测,以基质匹配曲线外标法定量。正离子采用CAPCELL PAK C18 MGⅢ-H色谱柱,流动相为0.05%甲酸乙腈和0.1%甲酸水;负离子采用ACQUITY UPLC BEH C18色谱柱,流动相为5 mmol/L乙酸铵和乙腈。结果表明,鸡蛋样品中的48种兽药残留在0.5~50 μg/kg浓度范围内线性关系良好,相关系数(r)为0.9952~1.0000。方法检出限为0.01~0.55 μg/kg,定量限为0.03~1.83 μg/kg,样品回收率在63.3%~111.4%之间,相对标准偏差小于10%(n=3)。本方法操作快速简单,重复性好,灵敏度较高,适用于鸡蛋中48种兽药残留的快速筛查检测。     

A multi-class multi-residue method for the analysis of polyether ionophores,tetracyclines and sulfonamides in multi-matrices of animal and aquaculture fish tissues by ultra-high performance liquid chromatography tandem mass spectrometry

ABSTRACT

An ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) based multiclass multi-residue method for the simultaneous analysis of 5 polyether ionophores, 4 tetracycline and 10 sulfonamides in animal and aquaculture fish tissues was developed and validated. Sample extraction and clean-up were based on a modified QuEChERS method. The method was validated using an in-house validation based on performance characteristics modified from Commission Decision 2002/657/EC. Both matrix effect and uncertainties associated with sample preparation and instrumental analysis were minimised by the use of matrix-matched calibrations. Recoveries of analytes were generally satisfactory and typically fell between 80% and 113%. The repeatability and intermediate reproducibility measured as relative standard deviations were in most cases less than 15% (n = 63). The decision limit (CCα) and detection capability (CCβ) ranged from 110.7 to 125.8 and 121.5 to 151.7 µg kg^?1 for tetracyclines, 113.4 to 118.3 and 116.8 to 126.5 µg kg^?1 for sulfonamides and 50.8 to 52.4 and 51.5 to 55.6 µg kg^?1 for polyether ionophores, respectively. The method displayed its fitness for purpose through satisfactory results obtained in international proficiency testing schemes. The method was applied to animal and aquaculture fish tissues obtained from different sources in South Africa. Polyether ionophores were predominantly detected in samples in the range 4.26–290.10 µg/kg. Oxytetracycline was found in one porcine liver sample; however, none of the targeted analytes were present above the detection limit in the aquaculture samples.     

Development of a new screening method for the detection of antibiotic residues in muscle tissues using liquid chromatography and high resolution mass spectrometry with a LC-LTQ-Orbitrap instrument

A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed for screening meat for a wide range of antibiotics used in veterinary medicine. Full-scan mode under high resolution mass spectral conditions using an LTQ-Orbitrap mass spectrometer with resolving power 60,000 full width at half maximum (FWHM) was applied for analysis of the samples. Samples were prepared using two extraction protocols prior to LC-HRMS analysis. The scope of the method focuses on screening the following main families of antibacterial veterinary drugs: penicillins, cephalosporins, sulfonamides, macrolides, tetracyclines, aminoglucosides and quinolones. Compounds were successfully identified in spiked samples from their accurate mass and LC retention times from the acquired full-scan chromatogram. Automated data processing using ToxId software allowed rapid treatment of the data. Analyses of muscle tissues from real samples collected from antibiotic-treated animals was carried out using the above methodology and antibiotic residues were identified unambiguously. Further analysis of the data for real samples allowed the identification of the targeted antibiotic residues but also non-targeted compounds, such as some of their metabolites.     

Optimization of microwave-assisted extraction with saponification (MAES) for the determination of polybrominated flame retardants in aquaculture samples

The efficiency of microwave-assisted extraction with saponification (MAES) for the determination of seven polybrominated flame retardants (polybrominated biphenyls, PBBs; and polybrominated diphenyl ethers, PBDEs) in aquaculture samples is described and compared with microwave-assisted extraction (MAE). Chemometric techniques based on experimental designs and desirability functions were used for simultaneous optimization of the operational parameters used in both MAES and MAE processes. Application of MAES to this group of contaminants in aquaculture samples, which had not been previously applied to this type of analytes, was shown to be superior to MAE in terms of extraction efficiency, extraction time and lipid content extracted from complex matrices (0.7% as against 18.0% for MAE extracts). PBBs and PBDEs were determined by gas chromatography with micro-electron capture detection (GC-muECD). The quantification limits for the analytes were 40-750 pg g(-1) (except for BB-15, which was 1.43 ng g(-1)). Precision for MAES-GC-muECD (%RSD < 11%) was significantly better than for MAE-GC-muECD (%RSD < 20%). The accuracy of both optimized methods was satisfactorily demonstrated by analysis of appropriate certified reference material (CRM), WMF-01.     

水产养殖投入品中药物残留检测方法研究及现状分析

目的建立不同类别水产养殖投入品中氯霉素类、孔雀石绿、己烯雌酚、磺胺类和喹诺酮类等5类14种药物残留的检测方法,用于调查和评价水产养殖投入品中药物残留现状。方法利用液相色谱-串联质谱仪和酶标仪,结合固相萃取等前处理净化方法,建立投入品中14种药物残留的检测技术,并用于实际水产养殖饲料样品、防治病药物和水质环境改良剂样品中药物残留含量的测定。结果饲料样品中5类药物检出率为62.5%,防治病药物中检出率为8.33%,水质环境改良剂中均未检出这5类药物残留。另外,检出药物品种主要为氟苯尼考和少量喹诺酮类,皆未超出国家限量。结论初步评估了投入品在水产养殖中的潜在风险:在水产养殖饲料样品中普遍添加国家限用药物,监管部门应加强其生产和使用管理;在防治病药物中需要注意药物的有效成分和比例;而目前水质改良剂相对比较安全,可根据需要放心使用。