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

Area of Science:

• Analytical Chemistry
• Biochemistry
• Pharmacology

Background:

• Quantification of phosphorodiamidate morpholino oligomers using LC-MS/MS faces challenges due to low sensitivity.
• Conventional solid-phase extraction methods exhibit poor recovery rates for these specific oligomers.
• A need exists for a more sensitive bioanalytical method with optimized pretreatment for accurate oligomer determination.

Purpose of the Study:

• To develop and validate a highly sensitive bioanalytical method for quantifying phosphorodiamidate morpholino oligomers in plasma.
• To improve upon existing pretreatment techniques for enhanced recovery and sensitivity.
• To establish a robust method applicable to various phosphorodiamidate morpholino oligomers.

Main Methods:

• Utilized protein precipitation (PPT) with methanol for sample pretreatment, achieving high recoveries (>90%) for viltolarsen.
• Employed reversed-phase liquid chromatography (RPLC) with gradient elution to minimize ion suppression.
• Combined PPT and RPLC for optimized LC-MS/MS analysis.

Main Results:

• Achieved a lower limit of quantification of 1 ng/mL for viltolarsen, significantly higher sensitivity than approved oligomers (10-20 ng/mL).
• Demonstrated excellent between-run accuracy (97.1%–109%) and precision (2.56%–10.5%).
• Successfully applied the method to another phosphorodiamidate morpholino oligomer, ψM23D (+07-18).

Conclusions:

• Established a simple, highly sensitive bioanalytical method for viltolarsen determination by optimizing RPLC and PPT.
• Validated the method's applicability to phosphorodiamidate morpholino oligomers with different sequences.
• The developed method offers improved sensitivity and recovery for oligomer quantification.