Development and validation of a volumetric absorptive microsampling assay for analysis of voriconazole and voriconazole N-oxide in human whole blood

Abstract

Voriconazole is a broad-spectrum antifungal triazole drug for the treatment of invasive fungal infections. It is extensively metabolized by hepatic drug metabolizing enzymes cytochrome (CYP) 2C19 and CYP3A4. Selective inhibition of intestinal CYP3A4 by grapefruit juice may increase the oral bioavailability of voriconazole in children. To test this hypothesis it is necessary to develop a sensitive assay for measuring voriconazole and its major metabolites in a small volume of blood. Mitra® devices from Neoteryx were employed to develop and validate the assay for the quantitation of voriconazole and voriconazole N-oxide. Mitra® devices utilize volumetric absorptive microsampling (VAMS™) technology that enables accurate and precise collection of a fixed volume (10 μL of blood), reducing or eliminating the volumetric blood hematocrit assay-bias associated with the dried blood spotting technique. We developed an ultra-performance liquid chromatographic method with tandem mass spectrometry detection for quantification of voriconazole and voriconazole N-oxide. Sample extraction of Mitra® devices, followed by reversed-phase chromatographic separation and selective detection using tandem mass spectrometry with a 4.00 minute runtime per sample was employed. Standard curves were linear between 10.0 to 10,000 ng/mL for both voriconazole and voriconazole N-oxide. Intra- and inter-day accuracy were within 87–102% and precision (CV) was <12% based on a 3-day validation study. Recoveries were ≥94 % for voriconazole and ≥87 % for voriconazole N-oxide. Voriconazole and voriconazole N-oxide were stable in human whole blood under assay conditions (19 h at room temperature and 24 h in autosampler). Voriconazole was stable for 1-month in dried microsamples under different conditions (4, −20 and −78 °C). This assay provides an efficient quantitation of voriconazole and voriconazole N-oxide and is ready to be implemented for the analysis of whole blood microsamples in a pediatric clinical trial investigating the impact of intestinal inhibition of CYP3A4 on voriconazole pharmacokinetics.