A simple, sensitive and selective method is described for the determination of itraconazole and its metabolite in human plasma, Miconazole as internal standard by using HTLCMS/ MS. The method consists of a online coupling of extraction with cyclone P (50mm x 0.5mm, 50μm) HTLC column by injecting 15μL sample and chromatographic separation is performed with C18 Reverse phase column using 90:10 Acetonitrile: 10mM Ammonium Formate Buffer (pH 6.8) as gradient mobile phase followed by quantification with Tandem Mass spectrometry (MS/MS) in selective reaction monitoring mode using Electro spray ionization mode (ESI) as an interface. The method was fully validated in terms of specificity, sensitivity, precision, accuracy and stability over a concentration range of 1 to 500g/ml for both Drug and its metabolite using 0.5ml of human plasma per assay. Stability assessment was also included. The total run time for sample analysis was 1.5 min and the lower limit of quantification was 1ng/mL for both drug and its metabolite. The validated method was applied in bioavailability and bioequivalence study.
