Extractability-mediated stability bias and hematocrit impact: High extraction recovery is critical to feasibility of...

Volumetric absorptive microsampling (VAMS), a new microsampling technique, was evaluated for its potential in supporting regulated bioanalysis. Our initial assessment with MK-0518 (raltegravir) using a direct extraction method resulted in 45-52% extraction recovery, significant hematocrit (Ht) related bias, and more importantly, unacceptable stability (>15% bias from nominal concentration) after 7-day storage. Our investigation suggested that the observed biases were not due to VAMS absorption, sampling techniques, lot-to-lot variability, matrix effect, and/or chemical stability of the compound, but rather the low extraction recovery. An effort to improve assay recovery led to a modified liquid-liquid extraction (LLE) method that demonstrated more consistent performance, minimal Ht impact (Ht ranged from 20 to 65%), and acceptable sample stability. The same strategy was successfully applied to another more hydrophilic model compound, MK-0431 (sitagliptin). These results suggest that the previously observed Ht effect and "instability" were in fact due to inconsistent extractability, and optimizing the extraction recovery to greater than 80% was critical to ensure VAMS performance. We recommend adding Ht-independent recovery as part of feasibility assessment to de-risk the long-term extractability-mediated stability bias before implementing VAMS in regulated bioanalysis.