A simple, sensitive and reproducible high performance liquid chromatography tandem mass spectrometric method was developed and validated for the determination of itraconazole and hydroxy itraconazole in human plasma using itraconazole-d9 and hydroxy itraconazole-d8, respectively, as internal standards. The precursor to product ion transitions of m/z 705.4 ®392.5, m/z 716.5 ® 402.6, m/z 721.3 ® 408.4 and m/z 729.4® 416.5 were used to detect and quantify itraconazole, hydroxy itraconazole, itraconazole-d9 and hydroxy itraconazole-d8 respectively. Sample preparation was carried out by solid phase extraction using hydrophiliclypophilic balance cartridges and the processed sample was chromatographed on Chromolith speed rod RP-18e (50 × 4.6 mm) column using 10 mM ammonium formate buffer (pH 4.0): methanol:: 20:80 v/v as the mobile phase. The weighed (1/x2) calibration curves were linear over the range of 0.301 to 151.655 ng/ml and 0.946 to 224.908 ng/ml for itraconazole and hydroxy itraconazole respectively. The RSD of intra-day and inter-day assay was ≤15%. Extraction procedure yielded a recovery of 52.07%, 53.73%, 49.68% and 50.16% for itraconazole, hydroxy itraconazole, itraconazole-d9 and hydroxy itraconazole-d8 respectively. The validated method was successfully employed for establishing pharmacokinetic parameters of two formulations of itraconazole 100 mg capsule in 14 healthy male Indian volunteers.
