Determination of emamectin residues in the tissues of Atlantic salmon (Salmo salar L.) using HPLC with fluorescence detection

An accurate, reliable, and reproducible assay for the determination of residual concentrations of emamectin B-1a in muscle, skin, and intact muscle/skin in natural proportions from Atlantic salmon treated with SCH 58854 (emamectin benzoate) is described. The determinative method was developed and validated using fortified control tissues at five levels over a range of 50-800 ng/g as well as tissues containing incurred levels in the same range. Incurred tissues were obtained from a metabolism study of [H-3]emamectin benzoate in Atlantic salmon. The assay employs processing of a tissue ethyl acetate extract on a propylsulfonic acid solid phase extraction cartridge, followed by derivatization with trifluoroacetic anhydride in the presence of N-methylimidazole. Following separation using reversed phase HPLC, the amount of derivatized emamectin B-1a is determined by fluorescence detection. The theoretical limits of detection were determined from the analysis of control tissue matrices to be 2.6, 3.3, and 3.8 ng/g as emamectin B-1a for muscle, skin, and intact muscle/skin, respectively. Likewise, the theoretical limits of quantitation (LOQ) were determined to be 6.9, 8.1, and 9.5 ng/g as emamectin Bla for muscle, skin, and intact muscle/skin, respectively. The lowest fortification level used for method validation was 50 ng/g,which served as the effective LOQ for the method. The overall percent recoveries (+/-% CV) were 94.4 +/- 6.89% (n = 25) for muscle, 88.4 +/- 5.35% (n = 25) for skin, and 88.0 +/- 3.73% for intact muscle/skin (n = 25). Accuracy, precision, linearity, selectivity, and ruggedness were demonstrated. The structure of the final fluorescent derivative of emamectin B-1a free base was identified by ESI(+)/LC-MS. The frozen storage stability of [H-3]emamectin Bla in tissues with incurred residues was demonstrated for similar to 15 months by radiometric analysis and for an additional similar to 13 months by fluorometric analysis for a total of similar to 28 months.