Quantification of several acidic drugs in equine serum using LC-MS-MS

The use of nonsteroidal antiinflammatory drugs in racehorses is allowed under most jurisdictions. Furosemide is administered to treat exercise-induced pulmonary hemorrhage. To help distinguish between therapeutic and illegal uses, racing regulatory bodies have set thresholds in serum for several drugs. The method for the simultaneous detection and quantification of furosemide, flunixin, ketoprofen, phenylbutazone and oxyphenbutazone using 500 µL of serum, and liquid extraction using diethyl ether : hexanes : dichloromethane followed by liquid chromatography tandem mass spectrometry quantitation, was developed and validated. Method validation included inter- and intraday precision and accuracy. Method validation also included bench-top, freeze-thaw, processed and long-term storage stability testing. For all stability testing, the compounds showed a breakdown of <15%. Inter- and intraday precision for all compounds was found to be within the acceptance interval of ±15% [±20% at the lower limit of quantitation (LLOQ)]. Accuracy data for all compounds were within the acceptance interval of ±15% (±20% at the LLOQ). Uncertainty was calculated using the simplified Guide to the Expression of Uncertainty in Measurement approach and was <30% for all drugs at 95% confidence level. The method was found to be both robust and accurate for all tested drugs.