Sampling only ten microliters of whole blood for the quantification of poorly soluble drugs: Itraconazole as case study

- Quantification of itraconazole using only 10 μL of whole blood.
- The method has been validated and the stability of itraconazole has been assessed.
- The presented method is suited to study the bioavailability of drug formulations.
- Volumetric absorptive microsampling diminishes the number of required study animals.

Nowadays in animal studies, it is important to comply with the so-called Three Rs rule by replacing or reducing the number of tested animals. Volumetric absorptive microsampling (VAMS) can be used to collect small quantities (10 or 20 μL) of whole blood, thereby limiting the amount of animals needed. In this study, a quantitative method was developed and subsequently validated for the poorly soluble drug itraconazole (ITZ) using VAMS and ultra-high performance liquid chromatography (UHPLC) coupled to tandem mass spectrometry (MS). A proof of concept study showed that the optimized method is applicable to test the bioavailability of drug formulations containing ITZ. Using VAMS, smaller blood volumes can be taken per sampling point (10-20 μL instead of the conventional 0.2-0.5 mL) avoiding the sacrifice of animals. Moreover, the same rats can be used to compare different drug formulations which strengthens the validity of the results. In long-term bioavailability studies, it is necessary to guarantee the stability of the tested drugs supported on VAMS devices. In this study, we show that ITZ was only stable for 24 h after collection with VAMS, but for at least two weeks by the storage of extracted samples at −80 °C.