Simultaneous determination of itraconazole and its CYP3A4-mediated metabolites including N-desalkyl itraconazole in human plasma using liquid chromatography-tandem mass spectrometry and its clinical application

Abstract

Background: Itraconazole (ITZ), a triazole antifungal agent, is metabolized to hydroxy-ITZ (OH-ITZ), keto-ITZ (KT-ITZ), and N-desalkyl ITZ (ND-ITZ) by cytochrome P450 3A4. The pharmacokinetics of ND-ITZ remain largely unknown due to the lack of an accurate and reliable determination method. This study aimed to develop a simultaneous determination method for ITZ and its three major metabolites including ND-ITZ in human plasma using isocratic liquid chromatography coupled to tandem mass spectrometry and then apply the method in a clinical setting. Methods: Plasma specimens were pretreated by protein precipitation with acetonitrile. The supernatant was separated on a 3-μm particle octadecyl silane column (75 × 2.0 mm I.D.) in an isocratic elution of acetonitrile and 5 mM ammonium acetate (pH 6.0) (57:43, v/v). The method was applied to 10 patients treated with oral ITZ. Results: The calibration curves of ITZ, OH-ITZ, KT-ITZ, and ND-ITZ were linear over the concentration ranges of 15-1500, 15-1500, 1-100, and 1-100 ng/mL, respectively. The pretreatment recoveries and matrix factors were 90.1-102.2% and 99.1-102.7%. Their intra- and inter-assay accuracies and imprecisions were 94.1-106.7% and 0.3-4.4%. The plasma concentrations of ITZ, OH-ITZ, KT-ITZ, and ND-ITZ 12 h after dosing ranged from 32.5-1127.1, 19.0-1166.7, 1.1-5.4, and 3.5-28.3 ng/mL, respectively, in immunocompromised patients. Conclusions: This study developed a simultaneous determination method for concentrations of ITZ and its three metabolites including ND-ITZ in a clinical setting.