Validated Liquid chromatography tandem mass spectrometric method for quantification of Itraconazole and Hydroxy traconazole in human plasma for pharmacokinetic study

By Anil. K. Patni, Tausif Monif, A. H. Khuroo, Ashok. K. Tiwary

 

Abstract

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 hydrophilic- lypophilic 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/x 2 ) 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.

Key Words : itraconazole, hydroxy itraconazole, liquid chromatography tandem mass
spectrometry, human plasma, pharmacokinetic study

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