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