Determination of Flunixin in Milk by Liquid Chromatography with Confirmation by Gas Chromatography/Mass Spectrometry and Selected Ion Monitoring

Abstract

A liquid chromatographic (LC) method was developed for the determination of flunixin (FNX) in raw bovine milk. The milk was acidified and mixed 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 EtOAc extract was washed with water at pH 3.5, the water was discarded, and the EtOAc layer was then extracted with 0.1M NaOH. The aqueous layer was drained, passed through a primed C18 solid-phase extraction (SPE) column, and eluted with EtOAc. The EtOAc layer was dried under N2, taken 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 C18 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 ultraviolet 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 ranged 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 lactating cow was dosed intravenously (2.2 mg/kg) with flunixin meglumine (Banamine) to generate incurred milk residues. FNX residues ranged from 7.34 ng/mL at 16 h postdose to 1.74 ng/mL at 24 h postdose. Both levels were obtained with additional beta-glucuronidase treatment (almost no incurred drug was detected at these low levels without the enzyme treatment).(ABSTRACT TRUNCATED AT 250 WORDS)