摘要
本研究以中华鳖中8种抗病毒药物(antiviral drugs,ATVs)及2种代谢物为研究对象,采用Wallelut Lipoclean磷脂去除小柱净化样品,建立了通过型固相萃取净化模式与超高效液相色谱-串联质谱法(UHPLC-MS/MS)相结合的快速检测方法。样品在37℃下经酸性磷酸酯酶酶解,1%乙酸乙腈提取,通过型固相萃取小柱净化,40℃下氮吹浓缩后采用UHPLC-MS/MS检测,多反应监测(MRM)模式采集数据,内标法定量。针对不同ATVs之间极性差异大的特点,实验优化了仪器参数和前处理条件,重点对色谱柱进行了筛选并排除了生物内源性物质尿苷对利巴韦林的分析干扰,同时讨论了前处理过程中提取液和净化小柱选择对提取效率和净化效果的影响。在最优实验条件下,10种目标物在一定质量浓度范围内线性关系良好,相关系数(r 2)为0.994~0.998,方法检出限(MDL)为0.05~1.1μg/kg,方法定量限(MQL)为0.18~3.8μg/kg。在低、中、高3个添加水平下10种目标物的平均回收率范围为82.5%~103%,相对标准偏差(RSD)为3.11%~12.1%。该方法操作简便、快速、灵敏,重复性好,可用于大批量样品的同时快速检测,并成功应用于省级水产品质量安全风险监测工作。
Antiviral drugs(ATVs)are not licensed for the treatment of viral diseases in food-producing animals in China.However,they are reportedly illegally used in the Chinese softshell turtle(Pelodiscus sinensis)aquaculture industry;hence,the use of ATVs has become a new aquatic food-safety problem.In this study,a method was developed for analyzing eight ATV residues(amantadine,rimantadine,lamivudine ribavirin,moroxydine,acyclovir,oseltamivir,and imiquimod)and two metabolites(1,2,4-triazole-3-carboxamide and oseltamivir acid)in Chinese softshell turtle muscle using pass-through solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS).The main factors that affect the sensitivity,precision,and accuracy of the method were optimized during sample pretreatment and instrumental analysis owing to the diverse chemical properties of the analytes.The performance of various chromatographic columns and mobile phases were investigated to eliminate interference from uridine during the detection of ribavirin.The proportion of acetonitrile in extration solution and solid-phase extraction column type were prioritized when optimizing extraction and purification parameter.Samples were hydrolyzed using phosphatase(acid type)in 6 mL ammonium acetate buffer,with optimum results obtained when enzymolysis was performed at 37℃,and pH 4.8 for 2 h,after which they were extracted with 14 mL of acetonitrile containing 1%acetic acid.The supernatant(2 mL)was purified using a Wallelut Lipoclean column operated in pass-through solid-phase-extraction mode.The sample was dried under nitrogen at 40℃and the residue was dissolved in 1 mL of methanol-acetonitrile(50∶50,v/v)solution.The sample was finally filtered through a 0.22-μm hydrophilic polytetrafluoroethylene syringe filter for UHPLC-MS/MS.The chromatographic separation of the analytes was performed on a hydrophilic interaction chromatographic column(HILIC-Z,100 mm×2.1 mm,2.7μm)via gradient elution using 2 mmol/L ammonium acetate aqueous solution(containing 0.1%formic acid)and acetonitrile as mobile phases A and B,respectively.The linear gradient program were as follows:0-1.0 min,3%A;1.0-5.0 min,3%A-20%A;5.0-6.0 min,20%A;6.0-6.5 min,20%A-50%A;6.5-8.0 min,50%A;8.0-8.5 min,50%A-3%A;8.5-12 min,3%A.The analytes were detected using a triple quadrupole mass spectrometer operating in positive-ion scanning and multiple-reaction monitoring(MRM)modes,with quantitative analysis performed using the internal standard method.Good linearities(r 2=0.994-0.998)were obtained for the ten target compounds at experimental concentrations under the optimal conditions.The method detection limits(MDLs)and method quantification limits(MQLs)were in the range of 0.05-1.1 and 0.18-3.8μg/kg,respectively.The average recoveries of the 10 target compounds ranged from 82.5%to 103%,with relative standard deviations(RSDs)in the range of 3.11%-12.1%at low,medium and high spiked levels.The developed method was applied to 67 Chinese softshell turtle samples obtained from a fish farm in Zhejiang Province.Ribavirin and metabolite were detected in real samples at total contents of 6.02-333.2μg/kg.The developed method is simple to operate,highly accurate,and suitable for use in risk-monitoring applications owing to its ability to rapidly determine eight ATV residues and two metabolites in Chinese softshell turtle samples.
作者
王亚琴
王坤
柯庆青
谭羽翀
王鼎南
李诗言
WANG Yaqin;WANG Kun;KE Qingqing;TAN Yuchong;WANG Dingnan;LI Shiyan(State Key Laboratory of Rice Biology and Breeding,Institute of Biotechnology,Zhejiang University,Hangzhou 310058,China;Zhejiang Fisheries Technology Extension Center,Hangzhou 310023,China)
出处
《色谱》
CAS
CSCD
北大核心
2024年第12期1164-1172,共9页
Chinese Journal of Chromatography
基金
国家自然科学基金(32372480)
浙江省“尖兵”“领雁”研发攻关计划(2023C02039)。
关键词
通过型固相萃取
亲水作用色谱
超高效液相色谱-串联质谱
抗病毒药物
代谢物
中华鳖
pass-through solid-phase extraction
hydrophilic interaction chromatography(HIC)
ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS)
antiviral drugs
metabolites
Chinese softshell turtle
作者简介
通讯联系人:王亚琴.Tel:(0571)88982680,E-mail:yaqinwang@zju.edu.cn;通讯联系人:李诗言,Tel:(0571)85029627,E-mail:jimmyuu1987@163.com.
