Quality by design-driven optimization of dried blood spot extraction for a bioanalytical LC-MS method

Dried Blood Spot (DBS) extraction satisfies several prerequisites for an effective sample preparation technique such as easy storage, shipment and logistics. Indeed, the DBS has been reported to be stable under ambient conditions and do not need any freezing; it is thereby cheap and hardly affected by biohazard contaminations. Moreover, this technique can be automated and thus included among high throughput extraction methods. Other evident advantages are the low sample consumption and microsampling, especially for the pharmaceutical and newborn clinical fields. In bioanalysis, liquid chromatography coupled to mass spectrometry in the multiple reaction monitoring mode (LC-MRM/MS) has been established as the technique of choice for the quantification of pharmaceuticals in biological matrices. Both adequate selectivity by detecting compound-specific mass transitions and short analysis time can be achieved by LC-MS techniques. The development of analytical methods based on the recent principles of Quality by Design (QbD) has been gaining popularity due to enhanced understanding of the effects of critical method parameters, greater flexibility and enhanced method performance. In this study, QbD was applied for investigating in a systematic way the effect of the different parameters involved in the extraction efficiency from DBS of protease inhibitors. This approach made it possible to identify the design space, a multidimensional region where the specified quality of the extraction was obtained with a chosen probability. The extent of DBS possibilities was widely exploited by QbD, which allowed a rational development and an in-depth understanding of the extraction method.