Automated liquid chromatography-tandem mass spectrometry method for the analysis of firocoxib in urine and plasma from horse and dog.

A rugged, sensitive and efficient liquid chromatography-tandem mass spectrometry method was developed and validated for the quantitative analysis of firocoxib in urine from 5 to 3000 ng/mL and in plasma from 1 to 3000 ng/mL. The method requires 200 microL of either plasma or urine and includes sample preparation in 96-well solid phase extraction (SPE) plates using a BIOMEK 2000 Laboratory Automated Workstation. Chromatographic separation of firocoxib from matrix interferences was achieved using isocratic reversed phase chromatography on a PHENOMENEX LUNA Phenyl-Hexyl column. The mobile phase was 45% acetonitrile and 55% of a 2 mM ammonium formate buffer. The method was accurate (88-107%) and precise (CV<12.2%) within and between sets. Extraction efficiencies (recovery)>93% were achieved and ionization efficiencies (due to matrix effects) were >72%. Extensive stability and ruggedness testing was also performed; therefore, the method can be used for pharmacokinetic studies as well as drug monitoring and screening. The data presented here is the first LC-MS/MS method for the quantitation of firocoxib in plasma (LLOQ of 1 ng/mL), a 25-fold improvement in sensitivity over the HPLC-UV method and the first quantitative method for firocoxib in urine (LLOQ of 5 ng/mL). Additionally the sample preparation process has been automated to improve efficiency.