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 plasma 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 standard (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 injected 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) and acetonitrile (60:40, v/v) was used in an isocratic condition, and ITZ was 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 4-1000 ng/ml. The mean predicted concentrations of the quality control (QC) samples deviated by less than 10% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay were within 8% relative standard deviation. Both ITZ and I.S. were stable in the injection solvent at 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 rats following administration of a single dose of itraconazole (15 mg/kg). (C) 2001 Elsevier Science B.V. All rights reserved.