Quantitation of itraconazole in rat heparinized plasma by liquid chromatography-mass spectrometry

A liquid chromatographic-mass spectrometric (LC-MS) assay was developed and validated for the determination of itraconazole (ITZ) in rat heparinized p lasma using reversed-phase HPLC combined with positive atmospheric pressure ionization (API) mass spectrometry. After protein precipitation of plasma samples (0.1 mi) with acetonitrile containing nefazodone as an internal sta ndard (I.S.), a 50-mul aliquot of the supernatant was mixed with 100 mul of 10 mM ammonium formate (pH 4.0). An aliquot of 25 mul of the mixture was i njected onto a BDS Hypersil C-18 column (50 x 2 mm; 3 mum) at a how-rate of 0.3 ml/min. The mobile phase comprising of 10 mM ammonium formate (pH 4) a nd acetonitrile (60:40, v/v) was used in an isocratic condition, and ITZ wa s detected in single ion monitoring (SIM) mode. Standard curves were linear (r(2) greater than or equal to 0.994) over the concentration range of 4-10 00 ng/ml. The mean predicted concentrations of the quality control (QC) sam ples deviated by less than 10% from the corresponding nominal values; the i ntra-assay and inter-assay precision of the assay were within 8% relative s tandard deviation. Both ITZ and I.S. were stable in the injection solvent a t room temperature for at least 24 h. The extraction recovery of ITZ was 96 %. The Validated assay was applied to a pharmacokinetic study of ITZ in rat s following administration of a single dose of itraconazole (15 mg/kg). (C) 2001 Elsevier Science B.V. All rights reserved.