Emamectin B1a

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 [(3)H]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 B(1a) 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 [(3)H]emamectin B(1a) in tissues with incurred residues was demonstrated for approximately 15 months by radiometric analysis and for an additional approximately 13 months by fluorometric analysis for a total of approximately 28 months.

An LC/ESI (positive-mode)-MS/MS method was developed for simultaneous quantification of 8 macrocyclic lactones (abamectin B1a, abamectin 8,9-Z isomer B1a, emamectin benzoate B1a, emamectin benzoate 8,9-Z isomer B1a, ivermectin, eprinomectin B1a, doramectin and moxidectin) in animal tissues, egg, milk and honey. The separation was achieved on a TSK-GEL ODS 100 V column (2.0 x 50 mm, 3 microm) with a mobile phase consisting of 0.1% formic acid in acetonitrile, and 0.1 mM ammonium formate-0.1% formic acid in water, at a flow rate of 0.2 mL/min with gradient elution. Linear calibration plots were obtained with high correlation coefficients (r=0.998-0.999). The LOQ and LOD ranged from 0.02-1.5 ng/mL and 0.1-5 ng/mL, respectively. Average recoveries were in the range of 70.8-117.1% with associated RSD values<15% (n=10) for repeatability and reproducibility. The spiking levels for recoveries and RSDs met the validation criteria for Japanese maximum residue limits (MRLs). Based on these results, the proposed analytical method has been proven to be highly efficient and suitable for routine determinations of macrocyclic lactones in animal food matrices.

A liquid chromatography (LC)/fluorescence procedure was validated for emamectin (EM B1a) and desmethylamino-emamectin (DMAEM B1a) residues in lobster tissue. They were extracted by shaking and sonicating with 1% ammonium acetate-methanol in the presence of sand. The extract was concentrated, partitioned with ethyl acetate, and cleaned up on a propylsulfonic cation exchange cartridge. The analytes were eluted from the cartridge with 5% ammonium hydroxide-methyl acetate, the eluate was concentrated, and the solvent was changed to dry 20% ethyl acetate-acetonitrile before derivatization with trifluoroacetic anhydride-N-methylimidizole. The products were analyzed by LC-fluorescence, and no interference [>limit of detection (LOD)] was detected in the control samples. Lobster tissues fortified with EM B1a and DMAEM B1a at 0.5, 5, 10, 25, and 50 ng/g gave overall mean recoveries of 96.7 +/- 12.4%, relative standard deviation (RSD) = 12.8% for EM B1 and 83.6 +/- 12.1%, RSD = 14.5% for DMAEM B1a. Regression analysis of the calibration data gave slopes of 0.90 (EM B1a) and 0.71 (DMAEM B1a) with an r2 = 0.99 for both compounds. The calculated LOD and limit of quantification (LOQ) for EM B1a were 1.10 and 3.32 ng/g, respectively, and for DMAEM B1a were 0.762 and 2.31 ng/g, respectively. Residues of EM B1a and DMAEM B1a in fortified lobster tissues stored at -20 degrees C showed that residues were stable for 10-12 months. No loss of EM B1a and DMAEM B1a residues was observed after 3 freeze/thaw cycles of fortified tissue in a 5-day period.