一步净化结合超高液相色谱串联质谱同时测定雅鱼中13类54种兽药残留

杨巧慧; 刘中良; 陈亚; 李霞雪; 张宇; 曾艳; 张建雄; 闻瑞琪; 兰韬

doi:10.13386/j.issn1002-0306.2023030052

一步净化结合超高液相色谱串联质谱同时测定雅鱼中13类54种兽药残留

One-step Purification Combined with Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry for the Determination of Residues of 54 Veterinary Drugs Belonging to 13 Classes in“Ya-Fish”

摘要

摘要: 目的：本研究建立了一种一步净化法结合液相色谱串联质谱（LC-MS/MS）同时检测雅鱼（Schizothorax prenanti）中13类54种兽药残留的快速检测方法。方法：向2.0 g样品中加入8 mL 0.2%甲酸-乙腈:水溶液（90:10，V/V）提取，冰水浴超声提取，LPAS一步净化。采用C₁₈柱，甲醇-0.1%甲酸/5 mmol/L乙酸铵溶液梯度洗脱，华体会（中国）多反应监测（dMRM），正/负离子模式，内标法定量，LC-MS/MS测定。以添加54种兽药的方式，对提取方式、提取体积、盐的加入、液相和质谱条件进行了优化，对方法性能（基质效应、方法检出限、方法定量限、准确度、精密度）等进行了验证。结果：54种兽药在0.1~50 μg/L范围内线性关系良好，决定系数（R²）≥0.992，方法检出限（LOD）为0.03~2.5 μg/kg之间，方法定量限（LOQ）为0.1~8.3 μg/kg之间，在低（添加量5~10 μg/kg）、中（添加量10~20 μg/kg）、高（添加量50 μg/kg）三个水平下54种药物的平均回收率为70.45%~118.1%，RSD为0.01%~9.9%。对50份养殖雅鱼样本应用该方法进行检测，7份样品有药物检出，恩诺沙星和达氟沙星各检出2次，磺胺甲噁唑检出4次，均低于定量限的定性检出。结论：该方法操作简单，一步净化，且方法检出限低、灵敏度高，准确度和精密度均较好，方法性能验证其符合多种兽药残留要求，可为大批量检测雅鱼中多种类兽药残留提供有力支持。

Abstract: Objective: In this study, a one-step purification method combined with liquid chromatography tandem mass spectrometry (LC-MS/MS) was established for the simultaneous detection of 54 veterinary drug residues belongs to 13 classes of ya-fish (Schizothorax prenati). Methods: 8 mL of 0.2% formic acid-acetonitrile:water solution (90:10, V/V) was added to 2.0 g sample and extracted by ultrasonication in an ice-water bath and cleaned up in one step by LPAS. The determination was performed on a C₁₈ column with a gradient elution of methanol-0.1% formic acid-5 mmol/L ammonium acetate solution, dynamic multiple reaction monitoring (dMRM), positive/negative ionization mode, and quantitative LC-MS/MS by internal standard method. By adding 54 veterinary drugs, the extraction method, extraction volume, salt addition, liquid phase and mass spectrometry conditions were optimized, and the method performance (matrix effect, detection limit, quantification limit, accuracy and precision) were verified. Results: The linear relationships between the 54 drugs and their concentrations were good within the range of 0.1~50 μg/L, coefficient of determination (R²) ≥0.992. The limits of detection (LODs) were between 0.03~2.5 μg/kg and the limits of quantification (LOQs) were 0.1~8.3 μg/kg. The average recoveries of the 54 drugs at low (5~10 μg/kg), medium (10~20 μg/kg) and the high level (50 μg/kg) ranged from 70.45% to 118.1%, with relative standard deviations (RSD) between 0.01% and 9.9%. This method was applied to detect the drug in 50 samples of cultured ya-fish, and drugs were detected in seven of them. Enrofloxacin and danofloxacin were each detected twice, and sulfamethoxazole was detected four times, all at levels below the limit of quantification. Conclusion: The method was simple to operate, one-step purification, and had low limits, high sensitivity, good accuracy and precision, which met the technical requirements of multi-veterinary drug residue detection, and can provide technical support for the rapid and efficient detection of various veterinary drug residues in large quantities of ya-fish.