Development and Validation of a Chromatographic and Electrophoretic Method for the Determination of Amikacin and Urea in Bronchial Epithelial Lining Fluid

Methods were developed to determine amikacin and urea in bronchial epithelial lining fluid of neonates. Urea was determined as ammonium by capillary electrophoresis in combination with capacitively coupled contactless conductivity detection (CE-(CD)-D-4). The ammonium was produced by enzymatic conversion of urea with urease enzyme. The background electrolyte (BGE) contained 30 mM malic acid, adjusted to pH 4.1 by l-arginine, and 10 mM 18-Crown-6. Lithium was used as internal standard. A +30 kV was applied on a fused silica capillary with 75 mu m internal diameter (ID) and total length of 65 cm (41 cm to (CD)-D-4 detector). The optimized separation was obtained in < 3 min with good linearity (R (2) = 0.9998) for urea concentrations ranging from 0.6 to 24 mg L-1. It also shows a good repeatability expressed by the RSD which is 0.7 and 1.3 % for intraday and interday precision, respectively. The LOD and LOQ are 0.14 and 0.5 mg L-1 respectively. As the CE-(CD)-D-4 method was not sensitive enough for amikacin, the latter was determined using liquid chromatography combined with pulsed electrochemical detection (LC-PED). The LOQ for amikacin base was found to be 0.06 mg L-1. In addition, the linearity was good (R (2) > 0.995) as well as the repeatability (RSD = 0.1 %, n = 3). For both the CE and LC method, no interference of matrix components was observed and the recoveries were found to be close to 100 %.