Bernardy, J.A., C. Vue, and M.P. Gaikowski. 2000. Validation of an HPLC method 
for oxytetracycline in coho salmon and northern pike fillet tissue. Submitted 
to the U.S. Food and Drug Administration, Center for Veterinary Medicine, 
July 10, 2000. 1273 p.

The oxytetracycline (OTC) label is currently restricted to specific diseases in 
salmonids and catfish. To support the extension and expansion of the label, an 
accepted analytical method for generating OTC residue depletion data in fillet 
tissue was validated with coho salmon and northern pike.
The accuracy, precision, and stability of the analytical method was determined 
using fortified coho salmon skin-on fillet tissue and northern pike skinless 
fillet tissue. The tissues were fortified with nominal concentrations of 4000, 
2000, 1000, 500, 10, and in the case of coho salmon, 13 ng/g OTC-base. The mean 
method recovery in coho salmon fillet tissue ranged from 90.8 to 110%. The 
precision ranged from 0.97 to 12 % relative standard deviation (RSD) and the 
method detection limit was 3.8 ng/g and quantitation limit 13 ng/g. In northern 
pike fillet tissue, the mean method recovery ranged from 89 to 98%. The 
precision ranged from 1.5 to 9.7 %RSD, the method detection limit was 2.8 ng/g, 
and quantitation limit 9.5 ng/g. Oxytetracycline was stable in the fortified 
tissues of both species for at least 56 days when stored at less than -70 °C. 
Oxytetracycline was also stable in fillet tissue extract of both species for at 
least 8 days under ambient temperature and fluorescent lighting.
Florfenicol was tested for interference with OTC-base chromatographic resolution 
and then added to the list of aquaculture chemicals that do not interfere with 
the HPLC analysis of OTC-base using this analytical method.
The analytical method’s precision was also determined using fillet tissues from 
coho salmon and northern pike that were offered OTC-medicated feed for 10 days 
prior to sampling. The precision with the biologically incurred residues, 2.0 
%RSD for coho salmon and 5.1% RSD for northern pike, was similar to the 
precision of the fortified samples. The concentration of OTC-base in the fillet 
tissue did not reach the tolerance limit in either species. An OTC residue 
depletion curve was estimated in skinless fillet tissue from northern pike that 
were offered 247 mg OTC-HCl per kg fish per day for ten days. The resulting 
depletion curve was monophasic with an elimination half life of 3.3 days.
The method is accurate, precise, sensitive and selective for OTC in fillet 
tissue of coho salmon and northern pike. Oxytetracycline is stable in fillet 
tissue and fillet tissue extracts. The method provides similar precision with 
biologically incurred and fortified OTC residues.