Determination of Ivermectin in Salmon Muscle Tissue by Liquid Chromatography with Fluorescence Detection

Abstract

A liquid chromatographic method was developed for determination of ivermectin B1a (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 C8 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 C18 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 × baseline noise) was 0.25 ppb extracted from tissue. Incurred tissues had an average concentration of 32 ppb, with an RSD of 3%.