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Three novel LC-UV methods for the determination of pentamidine (PTMD) and two of its new analogs in rat plasma are described. The chromatographic conditions (wavelength, acetonitrile percentage in the mobile phase, internal standard) were optimized to have an efficient selectivity. A pre-step of extraction was simultaneously developed for each compound. For PTMD, a solid phase extraction (SPE) with Oasis HLB cartridges was selected, while for the analogs we used protein precipitation with acetonitrile. SPE for PTMD gave excellent results in terms of extraction yield (99.7 +/- 2.8) whereas the recoveries for the analogs were not so high but were reproducible as well (64.6 +/- 2.6 and 36.8 +/- 1.6 for analog 1 and 2, respectively).

By means of a recent strategy based on accuracy profiles (beta-expectation tolerance interval), the methods were successfully validated. beta was fixed at 95% and the acceptability limits at +/- 15% as recommended by the FDA. The method was successfully validated for PTMD (29.6-586.54 ng/mL), analog 1(74.23-742.3 ng/mL) and analog 2 (178.12-890.6 ng/mL). The first concentration level tested was considered as the LLOQ(lower limit of quantification) for PTMD and analog 1 whereas for analog 2, the LLOQ was not the first level tested and was raised to 178.12 ng/ml. (C) 2010 Elsevier BM. All rights reserved.