Development of a Rapid Method for the Confirmatory Analysis of Flunixin Residue in Animal Tissues Using Liquid Chromatography–Tandem Mass Spectrometry

Flunixin is approved for use in veterinary medicine as a nonsteroidal anti-inflammatory agent. There is a need for the control of residues and development of methods to monitor its compliance with legislation. However, few methods have been reported for the analysis of flunixin in the animal liver, kidney, and fat. Therefore, the aim of this work was to develop a relatively rapid sample preparation and sensitive using liquid chromatography–tandem mass (LC-MS/MS) confirmatory method to detect flunixin residues in the liver, kidney, muscle, and fat of swine and chicken. After acid hydrolysis, the sample was extracted with ethyl acetate. The extract was finally evaporated to dryness and reconstituted in a water/methanol mixture, and determination was carried out by LC-MS/MS. Flunixin was detected using positive electrospray ionization in selected reaction monitoring (SRM). Estimated limit of quantification of the method was 0.5, 0.5, 0.25, and 0.05 μg/kg for the liver, kidney, muscle, and fat, respectively. The method was validated in animal tissues in terms of selectivity, linearity, trueness, precision, decision limit (CCα), and detection capability (CCβ). All the trueness values fell within a range between 73.6 and 84.8 %. Precision values for all levels of concentration tested showed excellent relative standard deviation (RSD < 15 %). The CCα and CCβ values have been established for each tissue. A relatively rapid and sensitive LC-MS/MS method for the quantitative determination of flunixin in different animal tissues was developed and validated. The method is suitable for monitoring the flunixin residues in animal tissues.