Quantification and validation of nine non-steroidal anti-inflammatory drugs in equine urine by gas chromatography mass spectrometry for doping control

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly implemented in the treatment of inflammation, pain and fever when used in therapeutic doses in human and veterinary medicine. The method for the simultaneous determination of nine non-steroidal anti-inflammatory drugs known as therapeutic prohibited substances in equine urine has been developed and fully validated according to the European Commission Decision 2002/657/EC and Association of Official Racing Chemists (AORC) criteria. The validation was in accordance with the International Screening Limits (ISL) for Naproxen, Flunixin, Ketoprofen, Diclofenac, Eltenac, Meclofenamic acid, Phenylbutazone, Vedaprofen and Carprofen in equine urine regulated by International Federation of Horseracing Authorities (IFHA). After basic hydrolysis, samples were extracted with C18 cartridge on the automated solid phase extraction. Several derivatization reagents were investigated and Trimethylphenylammonium hydroxide (TMAH)/Methanol (20/80, v/v) was selected. Analyses were carried out on GC-MS with Selected Ion Monitoring (SIM) mode but the method can be applied for a large number of analytes. Within laboratory reproducibility was not more than 12.8% (≤15%) and mean relative recoveries ranged from 91.1% to 104.1% for inter- and intra-day. The decision limits (CCα) and detection capabilities (CCβ) were evaluated at concentrations near the ISL for each therapeutic substance. The validation results demonstrated that the method is highly reproducible, easily applicable and selective for the analysis of some NSAIDs in equine urine that have not been previously published. Finally, the method was also applied to known positive samples.