A relative bioavailability study (RBA) of two phenytoin (PHT) formulations was conducted in rabbits, in order to compare the results obtained from different matrices (plasma and blood from dried blood spot (DBS) sampling) and different experimental designs (classic and block). The method was developed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) in plasma and blood samples. The different sample preparation techniques, plasma protein precipitation and DBS, were validated according to international requirements. The analytical method was validated with ranges 0.20–50.80 and 0.12–20.32 µg ml−1, r > 0.999 for plasma and blood, respectively. Accuracy and precision were within acceptance criteria for bioanalytical assay validation (< 15 for bias and CV% and < 20 for limit of quantification (LOQ)). PHT showed long-term stability, both for plasma and blood, and under refrigerated and room temperature conditions. Haematocrit values were measured during the validation process and RBA study. Finally, the pharmacokinetic parameters (Cmax, Tmax and AUC0–t) obtained from the RBA study were tested. Results were highly comparable for matrices and experimental designs. A matrix correlation higher than 0.975 and a ratio of (PHT blood) = 1.158 (PHT plasma) were obtained. The results obtained herein show that the use of classic experimental design and DBS sampling for animal pharmacokinetic studies should be encouraged as they could help to prevent the use of a large number of animals and also animal euthanasia. Finally, the combination of DBS sampling with LC-MS/MS technology showed to be an excellent tool not only for therapeutic drug monitoring but also for RBA studies.
Development and validation of a dried blood spot assay for the quantification of ribavirin using liquid chromatography coupled to mass spectrometry