Ultrasensitive Quantification of Poloxamer 188 in Adeno-Associated Virus Products by All-Ion Fragmentation MS/MS

Abstract Poloxamer 188 (P188; Pluronic F68) is a nonionic surfactant frequently used in biotherapeutics, particularly in adeno-associated virus (AAV) formulations, in which it is present at trace concentrations (0.001%−0.005%). However, its accurate quantification is difficult, because of its low abundance, frequently limited sample volumes, and the ionization variability of its PEO−PPO−PEO triblock. Here, we developed an all-ion fragmentation (AIF) workflow with 44 Da and 58 Da neutral-loss-triggered data dependent (dd)-MS2 that fragments all precursors and performs quantification via the polyethylene glycol (PEG) diagnostic ion (m/z 133.1), thereby decreasing spectral complexity and adduct variability. In contrast to the most sensitive reported method, LC−MRM, using a 20 μL injection volume, which achieved a dynamic range of 250−10,000 ng/mL (0.000025%−0.001%), our method achieved a 0.00002% LLOQ with 1 μL injections, thus providing an ∼20-fold sensitivity gain that enables replicate analysis and a dynamic range 2 orders of magnitude wider (0.00002%−0.1%). Our assay is vendor-, formulation-, and LC-buffer-agnostic, and achieves ±10% accuracy and ∼10% CVs across AAV-relevant levels. Significant P188 losses attributable to container adsorption were observed; incorporation of 50% acetonitrile restored recovery and is recommended for sample preparation.