DETERMINATION OF FLUNIXIN IN MILK BY LIQUID-CHROMATOGRAPHY WITH CONFIRMATION BY GAS-CHROMATOGRAPHY MASS-SPECTROMETRY AND SELECTED-ION MONITORING

A liquid chromatographic (LC) method was developed for the determinati on of flunixin (FNX) in raw bovine milk, The milk was acidified and mi xed with silica gel, and the mixture was packed into a chromatographic column, The column was defatted with water-saturated dichloromethane- hexane (30 + 70, v/v), and the analyte was eluted with EtOAc. The EtOA c extract was washed with water at pH 3.5, the water was discarded, an d the EtOAc layer was then extracted with 0.1M NaOH. The aqueous layer was drained, passed through a primed C-18 solid-phase extraction (SPE ) column, and eluted with EtOAc, The EtOAc layer was dried under Ng, t aken up in a solution of MeOH-(5 mM tetrabutylammonium [TBA]-H2PO4 + 2 mM NaOH) (50 + 50), sonicated, and filtered. FNX was determined by LC using a C-18 column (ODS Hypersil), a mobile phase mixture of 58% A ( MeOH) and 42% B (5 mM TBA-H2PO4 + 2 mM NaOH), and a diode-array ultrav iolet detector at 285 nm, FNX was determined in raw milk at 5 spiking levels (5, 10, 20, 40, and 80 ng drug/mL milk), Absolute recoveries ra nged from 69.6 to 74.4%, and relative standard deviations ranged from 1.1 to 6.9%, The limit of quantitation was 1.7 ng drug/mL milk, A lact ating cow was dosed intravenously (2.2 mg/kg) with flunixin meglumine (Banamine) to generate incurred milk residues, FNX residues ranged fro m 7.34 ng/mL at 16 h postdose to 1.74 ng/mL at 24 h postdose, Both lev els were obtained with additional beta-glucuronidase treatment (almost no incurred drug was detected at these low levels without the enzyme treatment), The presence of FNX in incurred milk was confirmed by gas chromatography/mass spectrometry with selected ion monitoring.