Quantification of PEG2000-DMG and TS-202, an ionic lipid, in rabbit plasma by LC-MS/MS: Application to complement C5 siRNA lipid nanoparticles.

A simple and robust assay was developed for the quantification of PEG2000-DMG and TS-202, key components of the lipid nanoparticle (LNP) formulation of E8001-LNP, which encapsulates small-interfering RNA targeting complement C5, in rabbit plasma. The assay utilizes high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). To prevent degradation of PEG2000-DMG, plasma samples were treated with a serine protease inhibitor prior to protein precipitation extraction. The analytes were separated on a reversed-phase column under gradient elution conditions. Detection was performed using selected reaction monitoring in positive ion mode, with mass transitions of m/z 780.8 → 698.9 for PEG2000-DMG and m/z 650.5 → 126.3 for TS-202. The quantification ranges were 0.05-5 µg/mL for PEG2000-DMG and 0.5-50 µg/mL for TS-202, with validated dilution integrity up to 100-fold. Accuracy and precision of quality control samples at five concentration levels met acceptance criteria, remaining within ±15%. Stability studies confirmed the bench-top, freeze-thaw, and long-term frozen stability of both analytes in rabbit plasma. Application to a toxicokinetic study in rabbits revealed that PEG2000-DMG was eliminated much faster than TS-202. The incurred sample reanalysis further confirmed the assay reproducibility.