Rapid Determination of Ketoconazole Level in Human Plasma by High Performance Liquid Chromatography

A simple, precise, and rapid high performance liquid chromatography (HPLC) method for the determination of ketoconazole level in human plasma using itraconazole as an internal standard (IS) was developed and validated. 0.25 ml plasma samples containing ketoconazole were mixed with 15 µg of the IS. After adding 0.25 ml acetonitrile, the mixture was vortexed for two minutes and then centrifuged for 10 minutes at 16000 rpm at room temperature. The clear supernatant was transferred into an auto-sampler vial, and 100 µl was injected into the HPLC system with a run time of 10 min. The compounds of interest were efficiently separated on 4.6 x 150 mm, Symmetry Shield TMRP18 5-µm steel column, using a Guard Pak pre-column module with    Radial-Pak C18 5-µm insert, and detected using Waters 2475 multi λ fluorescence detector with an the excitation and emission wavelengths set at 260 and 375 nm, respectively. The mobile phase consisted of 0.02 M potassium dihydrogen phosphate (pH = 6.0, adjusted with 0.1 M sodium hydroxide) and acetonitrile (40:60, v:v), and was delivered at a flow rate of 1.0 ml/min. No interference from blank plasma or commonly used drugs was observed; and the detection limit of ketoconazole was 0.1 µg/ml. The relationship between ketoconazole concentration in plasma and peak area ratio of ketoconazole /IS was linear (r2 ≥ 0.9979) in the range of 0.1– 20 µg/ml. Intra- and inter-day coefficients of variation (CV) were ≤ 8.1%  and  ≤ 9.7%, respectively, with corresponding biases of ≤ -13% and ≤ 0.9%, respectively. Mean extraction recovery of ketoconazole and IS were ≥ 85% and 92%, respectively. Using the method, ketoconazole was found to be stable for 48 hrs at -20°C (≥ 95%) in processed samples and for 8 weeks at -20°C (100%) in unprocessed samples. Key words: Ketoconazole, Itraconazole, Human plasma, HPL