羊肉组织中6种抗微生物药物残留检测的HPLC-MS/MS方法建立

Development of an HPLC-MS/MS method for detecting residues of six antimicrobial agents in lamb tissue

【目的】为了对羊肉及其副产品中抗微生物药物残留进行快速痕量检测,优化检测技术,建立羊可食组织中恩诺沙星、环丙沙星、氧氟沙星、庆大霉素、替米考星、泰乐菌素6种药物残留检测的高效液相色谱串联质谱法(HPLC-MS/MS)。【方法】对0.1%甲酸乙腈和0.1%甲酸甲醇2种不同药物提取液进行了对比;以动物性食品中氟喹诺酮类、氨基糖苷类和大环内酯类药物检测国家标准为参考,比较HLB和C_(18)2种固相萃取柱对6种目标药物的净化效果;同时,分别用0.1%甲酸水、乙腈、0.1%甲酸乙腈、甲醇、0.1%甲酸甲醇作为洗脱液对提取净化后的样品进行洗脱,对比交叉组合的提取和洗脱效果。将建立方法应用于实际样品检测,并与国家标准检测方法进行对比和统计分析。【结果】确定了以0.1%甲酸乙腈提取30 min,HLB固相萃取柱净化,0.1%甲酸甲醇洗脱时,恩诺沙星、环丙沙星、氧氟沙星、庆大霉素、替米考星、泰乐菌素6种药物质谱信息捕捉准确,色谱分离效果良好,在限定浓度范围内标准曲线回归系数R2均大于0.99,在5、20、50μg/kg 3个加标浓度下回收率为73.3%~114.0%,相对标准偏差(n=6)1.01%~13.01%。6种药物在羊3种可食组织中检出限(LOD)0.05~1.2μg/kg,定量限(LOQ)0.16~3.8μg/kg,均低于现行国家标准中规定的该类药物残留检出限。单因素方差分析结果显示,方法与国家标准检测方法在检测结果上差异不显著(P>0.05)。【结论】建立了羊可食组织中恩诺沙星、环丙沙星、氧氟沙星、庆大霉素、替米考星、泰乐菌素药物残留检测的高效液相色谱串联质谱法,该方法灵敏、准确、高效,可为羊肉及其副产品中抗微生物药物残留监测、筛选和精准识别提供技术支撑。

【Objective】This study was conducted to establish a high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)method in order to optimize the detection technology and improve accuracy and efficiency of detecting antimicrobial drug residues in mutton and its by-products,specifically enrofloxacin,ciprofloxacin,ofloxacin,gentamicin,tilmicosin,and tylosin in mutton and its byproducts.【Method】The study compared the extraction efficiency of 0.1%formic acid acetonitrile and 0.1%formic acid methanol.The purification effects of HLB and C_(18) solid-phase extraction columns on the six target antimicrobials were compared against national standards for detecting fluoroquinolones,aminoglycosides,and macrolides in animal food.Various eluents,including 0.1%formic acid water,acetonitrile,0.1%formic acid acetonitrile,methanol,and 0.1%formic acid methanol,were used to elute the extracted and purified samples,and their extraction and elution effects were compared.The established method was applied to the detection of actual samples and compared with the national standard.【Result】The results demonstrated that accurate information regarding enrofloxacin,ciprofloxacin,ofloxacin,gentamicin,tilmicosin,and tylosin could be captured by mass spectrometry using 0.1%formic acid acetonitrile extraction for 30 minutes,followed by washing with 0.1%formic acid methanol.The regression coefficients(R^(2))of the standard curves were all greater than 0.99 within the limited concentration range.The recovery rates ranged from 73.3%to 114.0%,and the relative standard deviations ranged from 1.01%to 13.01%at concentrations of 5,20,and 50μg/kg.The detection limits(LOD)and quantitative limits(LOQ)of the six drugs in three types of edible sheep tissues were found to be in the range of 0.05~1.2μg/kg and 0.16~3.8μg/kg,respectively,which were lower than the detection limits stipulated in current national standards.Single-factor analysis of variance indicated no significant difference between the established method and the national standard method(P>0.05).【Conclusion】A sensitive,accurate,and efficient HPLC-MS/MS method was established for the detection of enrofloxacin,ciprofloxacin,ofloxacin,gentamicin,tilmicosin,and tylosin residues in edible lamb tissues which could serve as a technical reference for monitoring,screening,and accurate identification of antimicrobial residues in mutton and its by-products.