Sensitive and selective quantification of total and free itraconazole and hydroxyitraconazole in human plasma using ultra-performance liquid chromatography coupled to tandem mass spectrometry

Objectives: Protein-free (unbound) drug concentrations have been reported to be better biomarker of pharmacodynamics compared with total drug concentrations. In this study, we developed and validated an assay for the quantification of total and free itraconazole and hydroxyitraconazole, a main metabolite with antifungal activity, in human plasma using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Design & methods: Plasma sample was ultra-filtrated for the measurement of free itraconazole and hydroxyitraconazole concentrations. The samples were prepared by solid phase extraction, and then subject to UPLC-MS/MS quantification. Results: The assay fulfilled the requirements of the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) guidelines for assay validation, with a lower limit of quantification of 10 ng/mL for total itraconazole and hydroxyitraconazole, and 0.1 and 0.5 ng/mL for free itraconazole and hydroxyitraconazole, respectively. Recovery rates of total itraconazole and hydroxyitraconazole from whole plasma ranged from 53.3% to 64.0%, and recovery rates of free itraconazole and hydroxyitraconazole from ultrafiltrated plasma ranged from 81.6% to 98.7%. Matrix effect varied between 79.1% and 109.4% for total itraconazole and hydroxyitraconazole, and between 81.3% and 99.7% for free itraconazole and hydroxyitraconazole. The assay was successfully applied to therapeutic drug monitoring of itraconazole in three patients with chronic progressive pulmonary aspergillosis or invasive pulmonary aspergillosis. Plasma free hydroxyitraconazole concentrations were 8.1-, 23.3-, and 51.1-fold higher than plasma free itraconazole concentrations in the three patients. Conclusions: A method for sensitive and selective quantification of plasma total and free itraconazole and hydroxyitraconazole concentrations was developed using UPLC-MS/MS. Free hydroxyitraconazole concentration may be most important in therapeutic drug monitoring of itraconazole.