Sampling only 10 μL of whole blood to study the bioavailability of itraconazole formulations in rats

Background
Volumetric absorptive microsampling (VAMS) offers a unique opportunity to collect small and accurate quantities of biological fluids. This sampling technique is of great interest for volume-limited samples and for the collection of multiple samples from the same animal. This serial sample collection may reduce the number of required study animals, thereby fulfilling the three Rs rule (refine, reduce, replace). Here, we demonstrate the applicability of VAMS to study the bioavailability of drug formulations in rats.

Methods
Four itraconazole-containing formulations were successively administered to rats with a wash-out period of one week. VAMS was used to collect 14 whole blood samples of only 10 μL each within a time frame of 48 hours after administration of the different drug formulations. Particular attention was paid to sample preparation and stability. The extraction of itraconazole and its main metabolite hydroxy-itraconazole was optimized to provide a high recovery and minimal matrix effects. A developed and validated LC–MS/MS method was used for the quantification of the two compounds. Pharmacokinetic profiles for the different formulations were constructed and compared.

Results
The stability of itraconazole and hydroxy-itraconazole in dried VAMS samples of whole rat blood could not be guaranteed for more than a day when the samples were stored at room temperature. However, samples were stable for at least two weeks when stored at -80°C after sample preparation.
Differences in pharmacokinetic profiles were observed for the tested drug formulations. Whole blood concentrations of itraconazole and its main metabolite were significantly higher after administration of three in-house produced formulations compared to concentrations obtained with a commercially available product. Moreover, these in vivo results could be partly related to in vitro dissolution rates of the various formulations.

Conclusions
VAMS is an attractive approach for bioavailability studies. Due to the low blood volumes per sampling point, the same rats can be used to compare various drug formulations. Therefore, the number of required animals can be drastically reduced. Moreover, this helps to suppress the inter-individual variability and strengthens the validity of results.