Abstract

Advancements in mRNA therapeutics have triggered the development of bioanalytical methods for quantification of oligonucleotides in biological matrices. Low sensitivity and specificity along with non-specific binding and low recoveries are drawbacks associated with bioanalysis of oligonucleotides. An LC-HRMS method was developed for quantification of unmodified oligonucleotides in human plasma samples. Development of a high-throughput sample preparation method with high recoveries >90% was achieved utilizing two LLE steps with phenol/chloroform and dichloroethane, requiring small sample volumes appropriate for preclinical studies. The developed method has overcome challenges on sensitivity due to signal division over multiple charge states and formation of adducts and specificity due to the use of a high resolving instrument. Another advantage was the use of the liquid handling workstation which allowed automation for a major part of the workflow. Automating the method with the use of the liquid handling workstation, facilitated the preparation and analysis of a large number of samples with high repeatability and reproducibility. The method was verified in the range of 15-3000 ng/mL with good precision (RSD)