Development and validation of a reversed-phase high-performance liquid chromatographic method for the determination of ethyl-3-(N-n-butyl-N-acetyl)aminopropionate in an insect repellent semi-solid formulation

A reversed-phase high-performance liquid chromatographic method with detection at 220 nm was developed and validated for the determination of ethyl-3-(N-n-butyl-N-acetyl)aminopropionate, IR 3535, in an insect repellent semi-solid product. A Hypersil ODS RP-C18 column (250×4.6 mm), 5 μm particle size, was equilibrated with a mobile phase consisted of water–acetonitrile (60:40, v/v). Its flow-rate was 1.0 ml/min. Excipients did not interfere with the determination of IR 3535 (Rs=8.663). Intra- and inter-day relative standard deviations for samples were not higher than 0.61 and 1.2%, respectively. Mean recovery was found not lower than 98.5% and not higher than 100.3%. The method of external standard was adopted. Calibration curves were linear in the concentration range between 1.0×10−6 and 5.0×10−4 M. Limits of detection and quantitation were 65 and 196 ng/ml, respectively.