Volumetric absorptive microsamplingcoupled with hybridization LC–MS/MS forquantitation of antisense oligonucleotides: supplementary materials

Background: Volumetric absorptive microsampling has emerged as a less invasive alternative to venous
sampling for small-molecule pharmacokinetic studies, but its application to novel therapeutics such as
antisense oligonucleotides (ASOs) is not well-established. Results: A workflow was developed using
Mitra microsampling coupled with hybridization LC–MS/MS for accurate determination of fomivirsen,
a 21-mer ASO, in human blood. Quantitative recovery was achieved regardless of blood hematocrit
level or microsample age by implementing impact-assisted extraction. A thorough method evaluation
confirmed sensitivity, linearity, precision/accuracy,matrix effect, metabolite interference and four months
of microsample stability. Conclusion: The combined impact-assisted extraction and hybridization LC–
MS/MS workflow demonstrated the successful quantitation of fomivirsen, establishing the validity and
applicability of the approach for ASO drug candidates.