High-performance liquid chromatography-based determination of nicarbazin excretion in waterfowl

A method for the high-performance liquid chromatography (HPLC) determination of nicarbazin uptake and excretion in ducks is presented. The method uses few clean-up steps and provides a rapid assessment of nicarbazin excretion by measuring the analyte 4,4′-dinitrocarbanalide (DNC). During method development the effect of extraction volume, number of extractions, mobile phase composition, column temperature, and injection volume were varied to optimize sensitivity and achieve as short a run time as possible. For our purposes, a 2×5.0 ml 1:1 dimethyl formamide (DMF):acetonitrile (ACN) extraction injected (40 ml) into an HPLC system equipped with a Keystone octadecylsilyl (ODS) C18 column and a UV variable wavelength detector (λ=347 nm) with a mobile phase of 60:40 (v/v) ACN–H2O, at a flow-rate of 1.0 ml/min at a column temperature of 35 °C provided adequate resolution and an acceptable total run time. Studies conducted during method development for inter-day recovery efficiencies for 0.46, 1.8 and 88.5 μg/g fortified samples (n=3) had mean recoveries of 91, 94 and 97% and intra-day (n=3) recoveries at the same fortification levels of 103, 94, and 92%. The method has been used successfully in excretion studies of nicarbazin in ducks.