High-performance liquid chromatographic determination of oxolinic acid residues in fish silage

A sensitive high-performance liquid chromatographic procedure was developed for the determination of oxolinic acid in fish silage. Oxolinic acid was extracted with a mixture of McIlvaine buffer (pH 3.6) and methanol (55:45) and re-extracted into dichloromethane. After successive clean-up by liquid-liquid partitioning, oxolinic acid was determined by HPLC with fluorimetric detection (exitation at 325 nm, emission at 360 nm). The analytical column was 3-microns MOS-Hypersil (150 x 4.6 mm I.D.) and the mobile phase contained (A) 0.025 M oxalic acid (pH 3.2)-acetonitrile-methanol-tetrahydrofuran (80:2.5:15:2.5, v/v) and (B) oxalic acid (pH 3.2)-acetonitrile-methanol-tetrahydrofuran (50:20:25:5), v/v) with the following elution profile: 0-5 min, linear gradient from 50 to 100% B; 5-10 min, isocratic at 100% B; 10.1-15 min, isocratic at 50% A-50% B. The calibration graph was linear over the concentration range studied (0.025-0.2 microgram/g). The limit of detection was 0.01 microgram/g (signal-to-noise ratio = 4).