| Abstract
| - An accurate, reliable, and reproducible assay for the determination of residual concentrations ofemamectin B1a in muscle, skin, and intact muscle/skin in natural proportions from Atlantic salmontreated with SCH 58854 (emamectin benzoate) is described. The determinative method was developedand validated using fortified control tissues at five levels over a range of 50−800 ng/g as well astissues containing incurred levels in the same range. Incurred tissues were obtained from ametabolism study of [3H]emamectin benzoate in Atlantic salmon. The assay employs processing ofa tissue ethyl acetate extract on a propylsulfonic acid solid phase extraction cartridge, followed byderivatization with trifluoroacetic anhydride in the presence of N-methylimidazole. Followingseparation using reversed phase HPLC, the amount of derivatized emamectin B1a is determined byfluorescence detection. The theoretical limits of detection were determined from the analysis of controltissue matrices to be 2.6, 3.3, and 3.8 ng/g as emamectin B1a for muscle, skin, and intact muscle/skin, respectively. Likewise, the theoretical limits of quantitation (LOQ) were determined to be6.9, 8.1, and 9.5 ng/g as emamectin B1a for muscle, skin, and intact muscle/skin, respectively. Thelowest fortification level used for method validation was 50 ng/g, which served as the effective LOQfor 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 fluorescentderivative of emamectin B1a free base was identified by ESI(+)/LC-MS. The frozen storage stabilityof [3H]emamectin B1a in tissues with incurred residues was demonstrated for ∼15 months byradiometric analysis and for an additional ∼13 months by fluorometric analysis for a total of ∼28months. Keywords: SCH 58854; avermectin; MK-244; emamectin; emamectin benzoate; emamectin B1a;salmon; analytical method
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