Determination Of Flunixin By HPLC And LC-MS/MS And Its Residual Depletion In Swine Tissues

Flunixin is a non-steroidal anti-inflammatory drug (NSAIDs), in veterinary medicine, it is used with meglumine as solubilizer as flunixin-meglumine(FM). Flunixin demonstrates potent inhibition of the cyclo-oxygenase system involved in the inflammatory pathway. Flunixin and its related products had been used in the domestic veterinary clinic, but the detection of flunixin residues in food of animal origin has not been reported. Therefore, in order to strengthen the monitoring of this drug, it is necessary to establish a method for determination of flunixin residues in swin tissues and study on the residues elimination of flunixin in swine.1. Determination of flunixin residues in swin tissues by HPLCFlunixin residues in swine tissues were extracted by acetonitrile with1%trichloroacetic acid, the extracts were defatted by n-hexane, applied to positive ion exchanging PCX cartridge. The sepration of flunixin was performed on a Hypersil C18column, and the mobile phase was methanol-0.05mol/mL phosphate (60:40, v/v, pH3.8adjusted by triethylamine). Under these chromatography conditions established in the assay, the concentration of flunixin had a good linear coefficient relation (R2>0.999) in the range of0.02～5μg/mL. The theoretical limit of detection was1,6,15, and15μg/kg for muscle, skin+fat, kidney and liver; the theoretical limit of quantitation was25μg/kg for muscle,10μg/kg for skin+fat,30μg/kg for kidney and liver. When spiked flunixin concentration in muscle were25,50,500and1000μg/kg, the recoveries were75.5%～93.5%; When spiked flunixin concentration in skin+fat were10,20,500and1000μg/kg, the recoveries were78.9%～97.4%; When spiked flunixin concentration in kidney and liver were30,60,500and1000μg/kg, the recoveries were73.6%～97.1%,71.9%～98.6%. The within-day and the between-day CV were2.28%～7.74%and3.39%～7.56%. The established method can be used for residue analysis of flunixin in swine tissues.2. Determination of flunixin residues in swin tissues by LC-MS/MSFlunixin residues in swine tissues were extracted by acetonitrile with1%trichloroacetic acid, the extracts were defatted by n-hexane, applied to positive ion exchanging PCX cartridge. The sepration of flunixin was performed on a Hypersil C18column, and a gradient elution with0.1%formic-acrtonitrile (containing0.2%formic) was used as mobile phase. Quantification was carried out by positive electrospray ionization (ESI+) and slected reaction monitoring (SRM) mod. Under these LC-MS/MS conditions established in the assay, the concentration of flunixin had a good linear coefficient relation (R2≥0.99) in the range of0.03～5μg/mL. The theoretical limit of detection was0.6μg/kg for muscle, skin+fat, kidney and liver; the theoretical limit of quantitation was1μg/kg for muscle, skin+fat, kidney and liver. When spiked flunixin concentration in muscle, skin+fat, kidney and liver were1,10,500and1000μg/kg, the recoveries were77%～91%,74%～94%,74%～91%and71%～98%. The within-day CV and the between-day CV were4.84%～8.64%and4.86%～10.03%. The established method can be used for residue analysis of flunixin in swine tissues.3. Study on the residues elimination of flunixin in swine25crossbred pigs were administered compound florfenicol injection twice interval for48h at2mg/kg b.w (flunixin counted). Five pigs per day were slaughtered at1,6,11,16,21days after the last administration. Flunixin in swine tissues were determinted by the established method above. The results showed the concentrations of flunixin in all tissues were (0.579±0.168) mg/kg,(0.447±0.190) mg/kg,(0.259±0.125) mg/kg,(0.050±0.003) mg/kg and (0.031±0.015) mg/kg for injection site, liver, kindy, muscle and skin+fat at day1, respectively.Flunixin were quickly eliminated in muscle and skin+fat, and slowly eliminated in injection site and kindy, liver was the slowest. This shows that the liver was the target organ of flunixin. The time when the concentrations of flunixin in muscle, skin+fat, kindy and liver were lower than LOD were6,6,11and16day. According to the residues of flunixin in swine liver and the EMEA rule of MRL, and the raw data were analysed with WT1.4program, the withdrawal period was7.51day in liver for flunixin, respectively; according to the residues of flunixin in swine liver and the FDA rule of MRL, and the raw data were analysed with WT1.4program, the withdrawal period was15.35day in liver for flunixin, respectively. Results of this experiment showed that the withdrawal period of flunixin in swine was16day.