Oxytetracycline (OTC)is a drug approved by the U.S. Food and Drug Administration 
to control ulcer disease, furunculosis and bacterial hemorrhagic septicemia in 
salmonids and to control bacterial hemorrhagic septicemia in channel catfish. 
Oxytetracycline is also approved to control gafkemia in lobsters and for marking 
salmonids for population assessment studies. The method currently approved for 
determining OTC in fish tissue is based on microbial inhibition and lacks 
sensitivity and specificity. The objective of our study was to validate a high 
performance liquid chromatography (HPLC) method for OTC in edible fillets from 
multiple species of fish for use in a bridging study with the microbial method. 
The HPLC method will be used to conduct residue depletion studies for the 
extension and expansion of the approved uses of OTC.
The method involved extracting OTC from homogenized fillet tissue by mixing 
tissue with a solution of citric acid, sodium phosphate, and ethylenediamine-
tetraacetic acid (EDTA). Particulate matter and precipitated proteins were 
removed with centrifugation. Oxytetracycline was extracted from the supernatant 
with phenyl solid phase extraction techniques and concentrations determined by 
isocratic reversed-phase HPLC with absorbance detection at 355 nm.
Fillet tissue was spiked at nominal concentrations of 20, 100, 1000, and 5000 
ng/ml. Mean recoveries of OTC from five species of fish ranged from 83.2% to 
98.4% and %RSD ranged from 0.9% to 11.4%. Method quantitation limits for the 
five species ranged from 6 ng/g to 23 ng/g. The method proved to be sensitive, 
specific, and robust for fish species from five phylogenetically diverse 
families.