Determination of Levamisole in Sheep Muscle Tissue by High-Performance Liquid Chromatography and Photo Diode Array Detection

Abstract

A high-performance liquid chromatographic method for the determination of levamisole (LVM) residues in sheep muscle tissue is described. LVM was extracted with ethyl acetate under alkaline conditions and cleanup was performed by liquid-liquid partition between organic-basic and organic-acid medium. Finally, levamisole was back extracted with chloroform carefully transferred into a clean glass vial and evaporated to dryness at 50 °C under a gentle stream of nitrogen. The remaining dry residue was dissolved in the mobile phase used, filtered and an aliquot was injected automatically into the chromatograph for analysis. Chromatography was performed on a Zorbax®SB-C18 column at 50 °C and detection by a PDA detector monitored at λmax 220 nm. The mobile phase was a mixture of 0.1 % trifluoroacetic acid (v/v) pH 2.0 and acetonitrile-methanol 3 : 2 (v/v) in a combination of 30 : 70 (v/v) and a flow rate of 1.0 mL min−1, delivered isocratically. This analytical method was validated by assessing recovery efficiency using spiked muscle tissue samples with standard solutions in methanol at four fortification levels of 1/2 MRL, 1 MRL, 2 MRL and 4 MRL and five times for each concentration (n = 5). Mean recovery (R%) achieved for muscle tissue was 75.65 ± 2.74% with an acceptable Relative Standard Deviation RSD% = 10.4. The same method was used also for the analysis of kidney, liver and fat (perirenal) and the recoveries found were 70.25 ± 1.07% (RSD% = 1.52), 72.37 ± 3.6% (RSD% = 4.97) and 69.44 ± 2.22% (RSD% = 3.19), respectively. The limit of detection (LOD) for muscle tissue was found to be 2.0 µg kg−1 and the limit of quantification (LOQ) 5.0 µg kg−1.