||Non-steroidal anti-inflammatory drugs (NSAIDs) |
|Drug Class Name
||Flunixin, NSAID, a nicotinic acid derivative|
||Determination of Flunixin in Cattle Liver by HPLC|
|Scope and Application
||This method can be used to screen for the presence of flunixin in bovine liver.|
||Homogenized tissue is hydrolized with HCI in tubes incubated on a heating block at 110 - 120 degrees celcius for 2 hours before cooling the mixture to ambient temperature. The pH of the hydrolysate is adjusted to 9.5 - 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 which is aliquoted for HPLC and LCMSMS and evaporated to dryness. This is capped and stored in a freezer at minus ten degrees celcius. The HPLC aliquot is later reconstituted in the mobile phase (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 HPLC UV. A C18 Hypersil analytical column is used for separation.
|Applicable Concentration Range
||Flunixin may be detected in bovine liver in the concentration range of 62.5 to 250 µg kg -1|
||See attached SOP|