Solid-phase extraction and liquid chromatographic quantitation of insect repellent N,N-diethyl-m-toluamide in plasma

A sensitive and reliable method based on solid-phase extraction and reversed-phase liquid chromatography was developed and validated for the quantitation of insect repellent N,N-diethyl-m-toluamide (DEET) in plasma. N,N-diethyl-2-phenylacetamide was used as internal standard in the extraction which employed C-18 solid-phase extraction cartridges. The wash solvent was 3 ml acetonitrile-ammonium acetate (pH 4.5; 0.03 M) (10:90, v/v), and the eluting solvent was 1 ml acetonitrile-ammonium acetate (pH 4.5; 0.03 M) (40:60, v/v). The eluent obtained from the extraction cartridge was directly analyzed on a reversed-phase C-8 column with UV detection at 220 nm. A clean chromatogram and high sensitivity were achieved at this wavelength. The mobile phase was acetonitrile-ammonium acetate (pH 4.5; 0.03 M) (36:64 v/v). The retention time was 7.9 min for the internal standard and 9.6 min for DEET when the mobile phase was delivered at 1.0 ml min(-1). The overall absolute recovery was 97.7% with a standard deviation (SD) of 3.9 (n = 9) for DEET and 100.2% with a SD of 3.4 (n = 3) for the internal standard. The limit of quantitation was found to be 15 ng ml(-1) with a relative standard deviation of 12%. For the analyses of DEET-spiked plasma samples with five replicates each at 50, 500 and 1500 ng ml(-1), the overall intra- and inter-day precisions were 5.7% and 5.5% respectively, and the overall intra- and inter-day accuracies were 2.0% and 2.4% respectively. The correlation coefficient for calibration plots in the concentration range 15-1500 ng ml(-1) was typically 0.999. The method was applicable to both dog and human plasma samples and was successfully used in pharmacokinetic studies of DEET in beagle dogs after intravenous bolus and topical routes of administration.