Microflow LC–MS/MS to improve sensitivity for antisense oligonucleotides bioanalysis: critical role of sample cleanness

Abstract

Background: Quantitative bioanalysis of antisense oligonucleotides (ASOs) is crucial to study their pharmacokinetic properties. An ultrasensitive bioanalytical method is often desired for quantifying low-concentration ASOs. Results: Effects of microflow LC and sample cleanness on sensitivity improvement of ASOs were evaluated. Sixfold sensitivity improvement of ASO-001 was achieved using microflow LC–MS/MS compared with conventional analytical flow method. Different sample extracts (hybridization, SPE and protein precipitation) were evaluated for sensitivity improvement by microflow LC. More sensitivity improvement was observed in the cleaner sample extract. Conclusion: Microflow LC increases sensitivity for ASO bioanalysis. The cleaner the sample extract, the better the sensitivity improvement. An ultrasensitive hybridization microflow LC–MS/MS method with lower limit of quantification of 0.100 ng/ml was developed and qualified for quantifying ASO-001 in plasma.