HPLC Assay of Levamisole and Abamectin in Sheep Plasma for Application to Pharmacokinetic Studies

Abstract

High performance liquid chromatography (HPLC) techniques were developed to quantify levamisole and abamectin in sheep plasma. UV detection (225 nm) for levamisole and fluorescence detection (excitation at 365 nm and emission at 470 nm) for abamectin were used. Separation was achieved on a C18 Prodigy ODS column with a mobile phase of phosphate (NaH₂PO₄ and Na₂HPO₄)‐acetonitrile (60∶40, v/v) (pH 7.5; 0.01M) for levamisole and methanol‐acetonitrile‐water (95∶3∶2, v/v/v) for abamectin. The retention times were 5.7 minutes for levamisole phosphate, 4.9 minutes for abamectin, 7.4 minutes internal standard (ivermectin). Calibration curves for levamisole phosphate and abamectin were linear over the range 0.05–10 µg/mL for levamisole phosphate and 0.25–20 ng/mL for abamectin, with the correlation coefficients for both drugs exceeding >0.999. The LOQ was achieved as the lowest point on the standard curve, 0.05 µg/mL for levamisole phosphate with the RSD 18.2% and 0.25 ng/mL for abamectin with the RSD 19.6%. The maximum intra‐day and inter‐day coefficients of variation were 9.1% and 15.0%, respectively, at 0.1 µg/mL (lowest concentration) for levamisole phosphate, and 11.9% and 19.3%, respectively, at 0.5 ng/mL (lowest concentration) for abamectin. Accuracies were 107.5% and 91.9% for levamisole and abamectin, respectively, at lowest concentrations of 0.1 µg/mL for levamisole phosphate and 1 ng/mL for abamectin. The recoveries from frozen and thawed plasma samples were 86.3% at 0.1 µg/mL for levamisole phosphate, and 105.8% at 2 ng/mL for abamectin. Both methods were successfully applied for analysis of levamisole and abamectin in plasma after subcutaneous injection to sheep of a formulation of medium chain mono‐ and diglyceride‐propylene glycol‐glycerol formal containing both levamisole phosphate and abamectin.

Keywords:

• High performance liquid chromatography
• Levamisole
• Abamectin
• Validation
• Plasma