Determination of Fluconazole in Children in Small Blood Volumes Using Volumetric Absorptive Microsampling (VAMS) and Isocratic High-Performance Liquid Chromatography–Ultraviolet (HPLC–UV) Detection

Objectives : A simple method for quantifying fluconazole in small blood volumes has been developed using volumetric absorptive microsampling (VAMS ® ) technology and isocratic high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. Methods : For sample collection, Mitra ® devices are used to keep the sample volume at 10 µL. For the quantitative determination of fluconazole, the Mitra ® samples are extracted using acetonitrile as the extraction agent, containing 2-(4-chlorophenyl)-1,3-bis(1,2,4-triazol-1-yl)propan-2-ol as the internal standard. A Synergi 4 μm Polar-RP 80 Å (150 × 2 mm) column forms the stationary phase, and a mixture of acetonitrile and phosphate buffer is the mobile phase. The UV detection is set at a wavelength of 210 nm. The therapeutic concentration range of 5 to 160 mg/L is covered, and the linear equation with 1/x 2 weighting is used to determine unknown samples. This method has been validated according to the current EMA and FDA guidelines for bioanalytical methods. Results : The validation data obtained after analysing whole blood samples (EDTA) showed within- and between-run accuracy between 94.4% and 115% and precision between 0.4% and 9.4%, respectively. A lower limit of quantification (LLOQ) of 5 mg/L was sufficient for therapeutic drug monitoring in paediatric patients receiving fluconazole as antifungal prophylaxis after haematopoietic cell transplantation. Conclusions : So far, 211 samples from 49 patients were successfully analysed, and concentrations between 5.84 mg/L and 107 mg/L were determined for whole blood Mitra ® samples. To our knowledge, this is the first application of VAMS ® technology using simple and cheap HPLC-UV quantification.