Measurement of Posaconazole, Itraconazole, and Hydroxyitraconazole in Plasma/Serum by High-Performance Liquid Chromatography With Fluorescence Detection

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Abstract

Background:

Itraconazole and posaconazole are used in the prevention and treatment of invasive fungal infections. However, the oral bioavailability of both compounds varies widely, and dose–serum concentration relationships are poorly defined for these analytes. The aim of this work was to develop and validate a simple assay that could be implemented in most laboratories for the purpose of therapeutic drug monitoring.

Methods:

Calibrators (n = 7) and internal quality control solutions (n = 3) were prepared in pooled human serum. Sample (100 μL), internal standard solution (25 μL), Tris solution (2 mol/L; pH 10.6), and extraction solvent (methyl tert-butyl ether, 600 μL) were vortex mixed and centrifuged. The solvent layer was removed and evaporated to dryness and the residue reconstituted in water:methanol (1 + 3, 50 μL). A portion (5 μL) of the reconstituted extract was analyzed using a 3-μm Gemini C6 phenyl column with fluorescence detection (excitation 260 nm, emission 350 nm). The method was used to measure itraconazole and hydroxyitraconazole, or posaconazole, in serum samples taken 1–2 hours before the next dose, from patients forming part of a study into management and diagnostic strategies for invasive aspergillosis.

Results:

Response was linear over the calibration ranges. Accuracy and imprecision were 92–111.4% and 3.2–13.4% (relative standard deviation), respectively. No interferences were noted. There was a good agreement with nominal values of each analyte in an external quality assessment scheme. In patients prescribed either 400 mg/d of itraconazole (n = 46) or 600–800 mg/d of posaconazole (n = 28) only 24% and 7% of samples, respectively, had serum itraconazole or posaconazole concentrations above the target threshold suggested in published guidelines.

Conclusions:

A simple, sensitive high-performance liquid chromatographic method has been developed for the analysis of itraconazole, hydroxyitraconazole, and posaconazole in serum/plasma. Few of the samples measured from patients participating in the clinical study attained concentrations of the drug/metabolite in serum that have been recommended for effective antifungal therapy.

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