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

Overview

• This study developed and validated a rapid and accurate method to detect 33 different non-steroidal anti-inflammatory drug (NSAID) residues in horse urine using a combination of QuEChERS sample preparation and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).
• The method is designed to monitor NSAID use in horses, especially during equestrian competitions, to ensure animal health and compliance with doping regulations.

Background and Importance

• NSAIDs are commonly used in horses to treat pain and inflammation caused by injuries during training and competitions.
• NSAIDs do not enhance a horse’s basic athletic ability but allow them to perform near maximum capacity by reducing pain, which can affect competition fairness and animal welfare.
• Monitoring NSAID residues is crucial in equestrian sports to prevent misuse and protect horse health, especially since some NSAIDs are prohibited during competition.

Method Development

• A rapid screening method was developed to detect 33 NSAIDs simultaneously in horse urine using QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) sample pretreatment combined with UPLC-MS/MS.
• Optimization involved refining the sample prep, chromatographic separation, and mass spectrometry detection conditions.

Sample Preparation

• 1 mL of horse urine was extracted with 5 mL acetonitrile, with 100 mg NaCl added to help separate the aqueous and organic layers (salting-out effect).
• The mixture was shaken for 3 minutes to ensure efficient extraction of NSAIDs.
• Extract purification used QuEChERS dispersive solid-phase extraction with:
  • 300 mg anhydrous magnesium sulfate (MgSO4)
  • 40 mg octadecylsilane-bonded silica (C18)
  • 40 mg primary secondary amine (PSA)
• After centrifugation, the supernatant was evaporated under nitrogen gas and reconstituted for analysis.

Chromatographic and Mass Spectrometry Conditions

• Chromatographic separation was done using an Agilent Poroshell 120 EC-C18 column (100 mm × 3.0 mm, 2.7 μm particle size).
• The mobile phases consisted of:
  • (A) 0.1% formic acid in water
  • (B) Methanol
• Gradient elution was carefully programmed over 17 minutes to separate all 33 NSAIDs efficiently.
• Detection used UPLC-MS/MS with dynamic multiple reaction monitoring (dMRM) and positive/negative ion switching to enhance sensitivity and specificity.
• Quantification was carried out using matrix-matched external standards to ensure accuracy in the presence of urine matrix components.

Method Performance

• All 33 NSAIDs showed good linearity across their target concentration ranges, with correlation coefficients above 0.99.
• Limits of detection (LOD) ranged from 0.1 to 1.0 μg/L, and limits of quantification (LOQ) ranged from 0.9 to 5.4 μg/L, indicating high sensitivity.
• Recovery rates for spiked urine samples were between 70.8% and 123.8%, confirming efficient extraction and low matrix interference.
• Precision was demonstrated by relative standard deviations (RSD) between 1.9% and 14.8% across the tested NSAIDs.

Application to Real Samples

• The method was applied to 10 actual horse urine samples collected during or near competition times.
• Three NSAIDs — ketorolac, lornoxicam, and naproxen — were detected in the ranges from below the LOQ up to 6.87 μg/L.
• According to the Federation Equestre Internationale (FEI) rules:
  • Ketorolac and naproxen are controlled substances prohibited during competitions but allowed outside these periods.
  • Lornoxicam is strictly banned at all times in horses.
• Detection of prohibited substances highlights the importance of effective monitoring for sporting fairness and horse welfare.

Conclusion and Significance

• The developed QuEChERS-UPLC-MS/MS method is simple, efficient, accurate, and capable of simultaneously quantifying 33 NSAIDs in horse urine.
• This method enhances the capacity to monitor NSAID use in equestrian sports, supporting anti-doping regulations and protecting equine health.
• The approach’s rapid turnaround and high sensitivity make it suitable for routine screening in veterinary and regulatory laboratories.