HPLC法测定鳗鱼中多种氟喹诺酮

采用高效液相色谱荧光检测法测定了鳗鱼中4种氟喹诺酮类(乳酸左氧氟沙星、诺氟沙星、乳酸环丙沙星和恩诺沙星) 药物残留量;样品用乙腈提取,正己烷脱脂,乙腈-水相进样,色谱分析采用J'sphere OD S-H 80 (150m m ×4.6m m ,4 m )柱,流动相为0.01m ol/L四丁基溴化胺(pH 3.0)∶ 乙腈(94∶6, μv/v),荧光检测波长为 ex= 280nm 、 em = 480nm ,乳酸左氧氟沙星、诺氟沙星、乳酸环丙沙星及恩诺 λ λ沙星的线性范围分别为0.05～1.2 g/m L、0.1～1.2 g/m L、0.05～1.2 g/m L和0.05～1.2 g/m L,检测限分别为 μ μ μ μ0.011 g/m L、0.023 g/m L、0.010 g/m L和0.031 g/m L。对空白样进行3个水平 (0.05m g/kg、0.1m g/kg和 μ μ μ μ0.2m g/kg)加标回收,回收率在86.5% ￣106.4% 之间。  

鸡肉中多种喹诺酮类兽药残留量的高效液相色谱测定研究

建立了一种测定鸡肉中氧氟沙星、诺氟沙星、环丙沙星、恩诺沙星兽药残留的高效液相色谱法。样品经乙腈提取，正己烷液一液分配去除蛋白和脂类物质，旋转蒸发除去乙腈，乙腈与水溶解残渣，过微孔滤膜，采用高效液相色谱荧光检测，外标法定量。氧氟沙星、诺氟沙星、环丙沙星、恩诺沙星在0．05～1．2μg／ml浓度范围内线性良好，相关系数为0．9997～0．9999。在0．05～1．0mg／kg浓度范围内，平均加标回收率在78．3％～101．8％之间，相对标准偏差为3．89％～12．43％。方法的最低检出限氧氟沙星为0．0037mg／kg、诺氟沙星为0．0037mg／kg、环丙沙星为0．0035mg／kg、恩诺沙星为0．0022mg／kg。该方法简便、快速，可满足鸡肉中多种唆诺酮类兽药残留量的检测。  

Antibodies to the quinolones and fluoroquinolones for the development of generic and specific immunoassays for detection of these residues in animal products

Several quinolone and fluoroquinolone haptens have been used to raise polyclonal antibodies exhibiting both specific and generic properties for these classes of antimicrobial compounds. The antisera have been assessed in rapid enzyme immunoassays (ELISAs) designed to exploit the specificities obtained. A direct generic ELISA for both the quinolones and fluoroquinolones has been developed that uses the cross-reactivity of an antibody raised against norfloxacin (1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid) linked to ovalbumin via a secondary amine group on the piperazinyl moiety to detect nine different drugs in these classes. Specific ELISAs to ciprofloxacin (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid), enrofloxacin (1-cyclopropyl-7-(4-ethyl-1-piperazinyl)-6-fluoro-1,4-dihydro-4-oxo-3-quinoline carboxylic acid), flumequin (9-fluoro-6,7-dihydro-5-methyl-1-oxo-1H,5H-benzo(ij)quinolizine-2-carboxylic acid) and nalidixic acid (1-ethyl-1,4-dihydro-7-methyl-4-oxo-1,8-naphthyridine-3-carboxylic acid) have also been developed with a high degree of specificity to the individual compounds. The assays measure drug residues in bovine milk and ovine kidney with an interassay relative standard deviation (s r ) of 10.5% or less and intra-assay s r of 11.2% or less. Sensitivity is less than 4 μg kg -1 for both the generic and specific assays for all but one of the compounds tested. (Pipemidic acid (8-ethyl-5,8-dihydro-5-oxo-2-(1-piperazinyl)pyrido(2,3-d)pyrimidine-6-carboxylic acid) is detectable at 6 μg kg -1 in kidney.)  

Qu ECh ERS EMR-Lipid结合LC-MS/MS测定鸡蛋中磺胺类和喹诺酮类药物残留

采用Qu ECh ERS EMR-Lipid技术处理样品,建立快速检测鸡蛋中14种磺胺类药物和16种喹诺酮类药物残留的液质联用检测方法。将鸡蛋样品用2%甲酸乙腈8 mL提取,再加入0.5 mL pH 7.0、0.1 mol/L的EDTA缓冲液震荡,用Qu ECh ERS EMR-Lipid快速样品提取技术净化,最后用0.2%甲酸溶液(A相)和乙腈溶液(B相)作为流动相进行梯度洗脱,采用电喷雾离子源的动态多反应监测模式(dynamic MRM)对30种磺胺类药物和喹诺酮类药物进行定性和定量分析。结果显示,30种抗生素添加水平在8~32μg/kg的回收率为50.09%~102.11%,相对标准偏差小于15.66%,方法检出限为0.00131~0.09717μg/kg。建立的鸡蛋中磺胺类药物和喹诺酮类药物残留检测的检测方法前处理简单易行,便于操作,方法精密度高,可以满足对鸡蛋中磺胺类药物和喹诺酮类药物快速、准确的检测要求,并适合大批量样品的准确定性和定量分析检测。  

不同前处理方法对猪组织中喹诺酮类兽药残留检测效果对比

采用超高效液相色谱-三重四极杆质谱和飞行时间质谱技术,比较EMR-L、Oasis PRi ME HLB和液液萃取法对猪组织中16种喹诺酮类兽药残留检测效果的影响。目标兽药以基质匹配工作曲线进行定量,在1.0~100.0 μg/L范围内线性关系良好,在5、10、20 μg/kg加标量条件下使用超高效液相色谱-串联质谱检测,EMR-L和Oasis PRi ME HLB方法处理的猪组织中加标回收率(71.7%~106.9%、70.7%~118.3%)、相对标准偏差(0.2%~16.8%、0.1%~17.8%)、定量限(0.13~0.56、0.27~1.36 μg/kg)和检出限(0.04~0.23、0.08~0.41 μg/kg)均满足方法学要求。针对不同基质成分样品,采用不同的前处理技术减少假阳性结果的产生,以适用于日益增长的动物源性食品兽药残留检测的需求。本研究采用的2种方法操作简便、灵敏度高、重复性好,满足对猪组织中多兽药残留的定性、定量分析。  

HPLC-MS-MS同位素内标法测定火腿中14 种喹诺酮类残留量

建立同时测定火腿中14 种喹诺酮类化合物高效液相色谱串联质谱检测方法。样品采用乙腈提取，乙腈饱和正己烷脱脂，经MAX固相萃取小柱净化，采用高效液相色谱串联质谱多反应监测正离子模式测定，使用同位素内标定量。14 种喹诺酮类检出限为0.6 μg/kg。方法平均回收率在70.0%～115.0%之间，相对标准偏差在1.2%～15.6%之间。该方法前处理简便快速、选择性好、灵敏度高，可作为日常的检测方法在实验室中使用。  

分散固相萃取-超高效液相色谱-串联质谱检测水产品中14 种喹诺酮类药物

采用基质固相分散的前处理技术，建立一种超高效液相色谱-串联质谱同时检测水产品中14 种喹诺酮类药物的分析方法。样品用5%甲酸-乙腈溶液提取和盐析剂盐析后加入N-丙基乙二胺和十八烷基键合硅胶吸附剂（C18）净化剂进行基质固相分散净化，氮吹复溶后经Waters ACQUITY UPLC BEH C18柱（50 mm×2.1 mm，1.7 μm）分离，以0.2%甲酸溶液和甲醇为流动相梯度洗脱，采用电喷雾离子源正离子多反应监测模式测定，外标法定量。14 种药物在0.50～20.0 μg/L质量浓度范围内呈良好线性，线性相关系数不小于0.995，方法检出限为0.4～1.0 μg/kg，定量限为1.0～3.0 μg/kg。14 种药物在3 个加标水平下的平均回收率为82%～90%，相对标准偏差（n=5）均小于11.0%。本方法简便快速、灵敏度高、实用性强，可作为水产品中14 种喹诺酮类药物残留的快速确证和定量分析。  

一种喹诺酮类药物的酶联免疫快速检测试剂盒的研制

利用酶联免疫技术研制了一种快速检测鱼肉中的喹诺酮类药物试剂盒,经过测试,该试剂盒对鱼肉样本中氧氟沙星、环丙沙星、氨氟沙星等3种喹诺酮类药物的检测限为1μg/kg,批内、批间相对标准偏差均小于10%;稳定性测试结果表明,试剂盒能够在4℃条件下保存12个月,37℃条件下保存7d。喹诺酮类药物单克隆抗体与氧氟沙星、环丙沙星、氨氟沙星的交叉反应率均为100%。该方法准确,可靠,使用简便,适合大量样品的现场检测。  

Characteristics of quinolone-resistant Escherichia coli isolated from bovine mastitis in China

Escherichia coli is the leading causative agent of bovine mastitis worldwide. Quinolone-resistant E. coli is becoming a potential threat to veterinary and public health. The aim of this study was to investigate the characteristics of quinolone-resistant E. coli isolated from bovine mastitis cases in China. Antimicrobial susceptibility of the isolates against 15 antimicrobial agents was determined by disc diffusion method. Phylogenetic grouping was detected by PCR. Extended-spectrum β-lactamase-producing isolates were determined by double-disc synergy test. In addition, the plasmid-mediated quinolone resistance (PMQR) and β-lactamase-encoding genes, as well as mutations of quinolone resistance-determining regions in GyrA, GyrB, ParC, and ParE, were measured by PCR and DNA sequencing. Overall, 75 (22.9%) out of 328 E. coli isolates were confirmed as ciprofloxacin-resistant from 2,954 mastitic milk samples. Phylogenetic group analysis showed that the majority of these strains belonged to phylogenetic group A (57.3%) and group B1 (24.0%). All the resistant isolates were identified as multidrug resistant, showing high resistance to cephalosporins and non-β-lactams. Forty-nine (65.3%) of the quinolone-resistant isolates were positive for PMQR genes; aac-(6')-Ib-cr was the most common PMQR determinant detected in 33 (44.0%) isolates. Eighteen (24.0%), 4 (5.3%), 3 (4.0%), and 1 (1.3%) of the quinolone-resistant isolates were harboring oqxA/B, qepA4, qnrS, and qnrB2, respectively. Additionally, 55 (73.3%) of the quinolone-resistant E. coli isolates were found to be extended-spectrum β-lactamase producers. The preponderant β-lactamase-encoding gene, bla_TEM, was detected in 44 (58.7%) isolates; bla_CTX-M, bla_CMY, and bla_SHV were found in 35 (46.7%), 22 (29.3%), and 2 (2.7%) isolates, respectively. Moreover, the most frequently identified substitutions were S83L/D87N or S83L in GyrA, detected in all of the quinolone-resistant isolates. Meanwhile, 74 (98.7%), 33 (44.0%), and 6 (8.0%) of the isolates were carrying substitutions S80I in ParC, S458A in ParE, and S492N in GyrB, respectively. All 58 (77.3%) isolates with a high level of ciprofloxacin resistance (>32 µg/mL) carried single or double mutations in GyrA combined with single mutation in ParC. To the best of our knowledge, this is the first report on the high occurrence of PMQR determinants and quinolone-determining resistant regions mutations in quinolone-resistant E. coli isolated from bovine mastitis in China.  

UPLC-MS/MS检测鸡蛋中22种喹诺酮类药物残留方法的研究

应用超高效液相色谱串联质谱建立鸡蛋中加雷沙星、曲伐沙星、莫西沙星、奥比沙星、吉米沙星、加替沙星、培氟沙星和环丙沙星等22种喹诺酮类药物残留检测方法。样品经过2%甲酸乙腈提取、正己烷分步去脂,Oasis HLB柱净化后,经Acquity UPLC BEH Shield RP18(100 mm×2.1 mm,1.7μm)分离,以甲醇和0.1%甲酸水溶液为流动相进行梯度洗脱,双通道MRM信号采集模式,22种喹诺酮类药物能在11 min完好分离,方法的最低定量限均低于1.0μg/kg,在2.0~100.0μg/L浓度范围内,22种喹诺酮类药物线性良好,相关系数均在0.99以上;通过2、10、20 g/kg三个浓度的加标回收实验表明,回收率为60.1%~96.2%,RSD%值为1.44%~11.1%。该方法相比现有国标检测药物种类多,新型药物多,对更全面筛查和确证鸡蛋中喹诺酮类药物残留,防范非法添加造成安全隐患具有一定的参考价值。