Simultaneous determination of polysorbate 20 and unbound polyethylene-glycol in protein solutions using new core–shell reversed phase column and condensation nucleation light scattering detection

A novel fast and sensitive method has been developed for the specific simultaneous determination of polysorbate 20 (Tween 20) and unbound polyethylene-glycol (PEG) from liquid formulations in the presence of proteins and excipients. The quantitative determination is based on a fast liquid chromatographic (HPLC) separation and condensation nucleation light scattering detection (CNLSD or NQAD™). The method uses a Kinetex core–shell column (100 mm × 3 mm, 2.6 μm) and methanol–water–trifluoroacetic acid mobile phase. The rapid HPLC-CNLSD method presented here is suitable for quantifying polysorbate 20 in the range of 10–60 μg/ml and unbound PEG in the range of 2–40 μg/ml in protein solutions within good manufacturing practices (GMP) of the pharmaceutical industry.