Determination of ivermectin in salmon muscle tissue by liquid chromatography with fluorescence detection

A liquid chromatographic method was developed for determination of ivermectin B 1a (IVR) extracted from raw fortified and incurred Atlantic salmon muscle tissues. The method was also used to determine fortified doramectin (DOR) in Atlantic salmon. Tissue extract was applied to a C 8 solid-phase extraction (SPE) column, followed by a silica SPE column. Residues in the eluate were treated with trifluoroacetic anhydride and methylimidazole to dehydrate the IVR molecule and form an aromatic fluorescent moiety with a trifluoroacetic ester. This product was subsequently treated with ammonium acetate in methanol to cleave the ester and convert the functional group back to a stable alcohol form. The analytes were determined by fluorescence with excitation at 272 nm and emission at 465 nm. A C 18 Hypersil column was used for analysis with a mobile phase of acetonitrile-water (90 + 10, v/v) and an oven temperature of 65°C. IVR and DOR were determined at 5 fortification levels (1, 5, 10, 20, and 40 ppb). Intra-assay absolute recoveries ranged from 75 to 89% for IVR and from 73 to 85% for DOR. Relative standard deviations (RSDs) were <7% in all cases. The limit of detection (3 x baseline noise) was 0.25 ppb extracted from tissue. Incurred tissues had an average concentration of 32 ppb, with an RSD of 3%.