A simple and highly sensitive LC-MS/MS bioanalytical method for phosphorodiamidate morpholino oligonucleotides in plasma

Background: LC-MS/MS is widely used for the quantification of phosphorodiamidate morpholino oligomers, but its low sensitivity is a problem. In addition, solid-phase extraction, a pretreatment method generally used for LC-MS/MS analysis, results in low recovery when applied to phosphorodiamidate morpholino oligomers. Therefore, a highly sensitive bioanalytical method for the determination of phosphorodiamidate morpholino oligomers using LC-MS/MS with improved pretreatment is desired. Results: Protein precipitation was applied to LC-MS/MS analysis for viltolarsen, as a typical phosphorodiamidate morpholino oligomer, in plasma, and PPT with MeOH achieved high recoveries (>90%). Reversed-phase liquid chromatography (RPLC) with gradient elution improved ion suppression compared to hydrophilic interaction liquid chromatography. A combination of PPT and RPLC achieved higher sensitivity (lower limit of quantification, 1 ng/mL) than those of approved phosphorodiamidate morpholino oligomers (10 to 20 ng/mL in their clinical reviews), and the between-run accuracy and precision ranged from 97.1% to 109% and 2.56% to 10.5%, respectively. This method was also shown to be applicable to another phosphorodiamidate morpholino oligomer, ψM23D (+07-18). Conclusion: A simple and highly sensitive bioanalytical method for the determination of viltolarsen was established by optimizing RPLC and PPT, and the method was successfully applied to a phosphorodiamidate morpholino oligomers with a different sequence.