色谱

色谱 ›› 2026, Vol. 44 ›› Issue (9): 1028-1037.DOI: 10.3724/SP.J.1123.2025.08014

• 研究论文 • 上一篇    下一篇

QuEChERS-超高效液相色谱-串联质谱法测定马尿中33种非甾体抗炎药残留

张晓男1,2, 马智超1,2, 刘安1, 黄渊3,*(), 闵雪红1,2, 刘梅2, 谢正齐1, 魏岩松1   

  1. 1.武汉商学院体育学院·国际马术学院，湖北 武汉 430056
    2.运动马检测及应用转化湖北省工程研究中心，湖北 武汉 430056
    3.华中科技大学同济医学院附属武汉儿童医院药学部，湖北 武汉 430016
  • 收稿日期:2025-08-20 出版日期:2026-09-08 发布日期:2026-09-17
  • 通讯作者: *E-mail：1762872223@qq.com.
  • 基金资助:
    湖北省地方标准制修订项目(T-Z-05-2024368);武汉商学院校级科研项目(2024KY019);武汉商学院学术团队项目(2024TD021)

Determination of 33 non-steroidal anti-inflammatory drug residues in horse urine by QuEChERS-ultra performance liquid chromatography-tandem mass spectrometry

ZHANG Xiaonan1,2, MA Zhichao1,2, LIU An1, HUANG Yuan3,*(), MIN Xuehong1,2, LIU Mei2, XIE Zhengqi1, WEI Yansong1   

  1. 1.School of Physical Education and National Equestrian Academy，Wuhan Business University，Wuhan 430056，China
    2.Hubei Provincial Engineering Research Center of Racing Horse Detection and Application Transformation，Wuhan 430056，China
    3.Department of Pharmacy，Wuhan Children’s Hospital，Tongji Medical College，Huazhong University of Science & Technology，Wuhan 430016，China
  • Received:2025-08-20 Online:2026-09-08 Published:2026-09-17
  • Supported by:
    Hubei Local Standard Formulation and Revision Project(T-Z-05-2024368);Wuhan Business University Research Project(2024KY019);Wuhan Business University Academic Team Project(2024TD021)

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摘要：

为有效监测马匹尿液中多种非甾体抗炎药的残留，为维护马术（赛马）运动的公平性和纯洁性提供重要的技术手段，实验利用QuEChERS前处理方法结合超高效液相色谱-三重四极杆质谱技术建立了马尿中33种非甾体抗炎药的分析方法，并对QuEChERS前处理中相关参数、色谱条件和质谱条件进行了优化。具体步骤：马尿样品经乙腈提取，NaCl为盐析剂，无水MgSO4为除水剂，十八烷基硅烷键合硅胶（C18）和N-丙基乙二胺（PSA）为净化吸附剂，经QuEChERS净化，离心后，上清液氮吹复溶，待测分析物经Agilent Poroshell 120 EC-C18柱（100 mm×3.0 mm，2.7 μm）分离，以0.1%甲酸水和甲醇为流动相，梯度洗脱，正、负离子切换动态多反应监测（DMRM）模式检测，基质匹配标准曲线外标法定量。结果表明，该方法可用于马尿中33种非甾体抗炎药同时快速测定，检出限（LOD）为0.1~1.0 μg/L，定量限（LOQ）为0.9~5.4 μg/L，在0.9~220 μg/L范围内线性关系良好，相关系数均大于0.99。在LOQ、20 μg/L和200 μg/L 3个添加水平下，马尿中33种非甾体抗炎药的平均回收率为70.8%~123.8%，相对标准偏差（RSD）为1.9%~14.8%。采用本方法对10份马尿样品进行检测，共检出酮咯酸、氯诺昔康、萘普生3种禁用物质，含量均≤6.87 μg/L。酮咯酸和萘普生为受限物质，休赛期允许使用；氯诺昔康为严禁物质，不允许马匹使用。该方法简单、高效、准确，适用于马尿中33种非甾体抗炎药残留的同时测定。

关键词: QuEChERS, 超高效液相色谱-串联质谱, 非甾体抗炎药, 马尿, 残留

Abstract:

Non-steroidal anti-inflammatory drugs （NSAIDs） are often used to treat horses’ injuries and pain due to long-term training and competitions. In equestrian competitions， NSAIDs do not affect the upper limit of a horse’s athletic ability， but they can enable the horse to perform close to its maximum capacity by reducing inflammation and pain. This not only affects the competition results， but also fails to ensure the health and welfare of the horses. Therefore， it has become crucial to effectively monitor and control NSAIDs residues during equestrian competition. In this study， a rapid screening and determination method for 33 NSAIDs in horse urine was established using QuEChERS pretreatment combined with ultra performance liquid chromatography-tandem mass spectrometry （QuEChERS-UPLC-MS/MS）. The pretreatment process， chromatographic conditions， and mass spectrometric conditions were also optimized. In brief， the target analytes in 1 mL of horse urine samples were extracted with 5 mL of acetonitrile. During extraction， 100 mg of NaCl was added as a salting-out agent， and the mixture was shaken for 3 min. Subsequently， the extract was purified using a QuEChERS method with 300 mg of anhydrous MgSO4， 40 mg of octadecylsilane-bonded silica （C18）， and 40 mg of primary secondary amine （PSA） as sorbents. After centrifugation， the supernatant was dried under nitrogen and reconstituted. The 33 NSAIDs were separated on an Agilent Poroshell 120 EC-C18 analytical chromatographic column （100 mm×3.0 mm，2.7 μm） with gradient elution using （A） 0.1% formic acid aqueous solution and （B） methanol solution as the mobile phases. The gradient elution program was as follows： 0–1 min， 30%B； 1–3 min， 30%B–60%B； 3–12 min， 60%B–85%B； 12–12.1 min， 85%B–95%B； 12.1–14 min， 95%B； 14–14.1 min， 95%B–30%B； 14.1–17 min， 30%B. Then， the target analytes were determined by UPLC-MS/MS in dynamic multiple reaction monitoring （dMRM） mode with positive/negative ion switching. Quantification was performed using the matrix-matched external standard method. The results showed that the method can rapidly and simultaneously determine the 33 NSAIDs in horse urine. The analytes had good linear relationships within their respective ranges， with correlation coefficients of >0.99. The limits of detection （LODs） and quantification （LOQs） were 0.1–1.0 μg/L and 0.9–5.4 μg/L， respectively. The recoveries of the 33 NSAIDs in horse urine were between 70.8% and 123.8% at three spiked levels of LOQ， 20 μg/L and 200 μg/L. The relative standard deviations of all targeted compounds ranged from 1.9% to 14.8%. The method was applied to 10 actual horse urine samples. The results revealed the presence of three NSAIDs， including ketorolac， lornoxicam and naproxen， with contents ranging from < LOQ to 6.87 μg/L. According to the Equine Prohibited Substances List published by the Federation Equestre Internationale （FEI）， ketorolac and naproxen are controlled medications. They are prohibited for use during competition period， as they may mask symptoms and aggravate clinical conditions. However， they are permitted for use during non-competition times. Lornoxicam is a banned substance and is strictly prohibited for use in horses at any time. The proposed method is simple， efficient and accurate， and is suitable for the simultaneous determination of the 33 NSAIDs residues in horse urine samples.

Key words: QuEChERS, ultra performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）, non-steroidal anti-inflammatory drugs （NSAIDs）, horse urine, residues

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