Measurement of posaconazole, itraconazole, and hydroxyitraconazole in plasma/serum by high-performance liquid chromatography with fluorescence detection

• Published in: Therapeutic drug monitoring Vol. 33; no. 6; p. 735
• Main Authors: Buckner, Sara L, Ceesay, M Mansour, Pagliuca, Antonio, Morgan, Phillip E, Flanagan, Robert J
• Format: Journal Article
• Language: English
• Published: United States 01.12.2011
• Subjects: Adult; Aged; Antifungal Agents - administration & dosage; Antifungal Agents - blood; Calibration; Chromatography, High Pressure Liquid; Cohort Studies; Dose-Response Relationship, Drug; Drug Monitoring - methods; Female; Humans; Invasive Pulmonary Aspergillosis - blood; Invasive Pulmonary Aspergillosis - complications; Invasive Pulmonary Aspergillosis - drug therapy; Itraconazole - administration & dosage; Itraconazole - analogs & derivatives; Itraconazole - blood; Limit of Detection; Male; Middle Aged; Neutropenia - complications; Reproducibility of Results; Spectrometry, Fluorescence; Triazoles - administration & dosage; Triazoles - blood; Young Adult
• ISSN: 1536-3694
• DOI: 10.1097/FTD.0b013e3182381bb1

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. 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. 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. 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.