Veterinary Drugs Residue Methods


Category Non-steroidal anti-inflammatory drugs (NSAIDs)
Drug Class Name Nonsteroidal anti- inflammatory drug (NSAID)
Method Title Confirmation of Flunixin Residues in Bovine Liver by HPLC/ESI-MS/MS
Method Date 2003/03/24
Method Type Confirmatory
Scope and Application The method is useful for confirming the presence of flunixin in bovine liver.
Method Summary Homogenized tissue is hydrolized with HCl in tubes incubated on a heating block at 110 to 120 degrees celcius for 2 hours before cooling the mixture to ambient temperature. The pH of the hydrolysate is adjusted to 9.5 to 9.70 using NaOH. Sodium chloride is added to the tubes followed by ethyl acetate prior to extraction by repeated centrifugation. The ethyl acetate extract is evaporated to dryness, reconstituted in hexane and mixed by vortex prior to cleane-up using a silica cartridge. The centrifuge tube is rinsed with 30 percent ethyl acetate,hexane and the contents loaded onto the cleanup column as well. Ethyl acetate is further used to wash the column contents into waste. The tubes are further rinsed twice with sodium phosphate and both eluates combined. After refrigerating the contents overnight at 2-8 degrees celcius, the aqueous sodium phosphate solution is further cleaned-up using C18 cartridge material. After loading the extract, this cartridge is washed with water and 5 percent MeOH,H2O (v,v) into waste before eluting the contents from the cartridge with 40 percent MeOH,H2O (v,v). This eluate is loaded onto SCX cartridges for final cleanup. After washing the column with methanol, the analyte is eluted using 10 percent NH4OH,MeOH. This solution is evaporated to dryness and the residue reconstituted in methanol. An aliquot for LCMSMS (the rest may be used for HPLC analysis) is evaporated to dryness and after storage (capped) in a freezer at minus ten degree celcius, the aliquot is later reconstituted in the mobile phase of 0.005 M Tetrabutyl Ammonium Phosphate,Methanol (46.5,53.3 v,v) and pressed through a 0.2 micron filter ready for analysis by LC,ESI-MS,MS. A Zorbax Rx C8 analytical column is used for separation.
Applicable Concentration Range The presence of flunixin in bovine liver may be confirmed using the concentration level of 125 µg kg -1.
QC Requirements See attached SOP
Method Source USDA-FSIS
Method SOP PDF icon SOP