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.