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A spectrophotometric method was developed to quantify low polysorbate (PS) levels in biopharmaceutical formulations containing high protein concentrations. In the method, Oasis HLB solid phase extraction (SPE) cartridge was used to extract PS from high protein concentration formulations. After loading a sample, the cartridge was washed with 4M guanidine HCl and 10% (v/v) methanol, and the retained PS was eluted by acetonitrile. Following the evaporation of acetonitrile, aqueous cobalt-thiocyanate reagent was added to react with the polyoxyethylene oxide chain of polysorbates to form a blue colored PS-cobaltothiocyante complex. This colored complex was then extracted into methylene chloride and measured spectrophotometrically at 620 nm. The method performance was evaluated on three products containing 30-40 mg L-1 PS-20 and PS-80 in <= 70 g L-1 protein formulations. The method was specific (no matrix interference identified in three types of protein formulations), sensitive (quantitation limit of 10 mg L-1 PS) and robust with good precision (relative standard deviation <= 6.4%) and accuracy (spike recoveries from 95% to 101%). The linear range of the method for both PS-20 and PS-80 was 10 to 80 mg L-1 PS. By diluting samples with 6 M guanidine HCl and/or using different methylene chloride volumes to extract the colored complexes of standards and samples, the method could accurately and precisely quantify 40 mg L-1 PS in up to 300 g L-1 protein formulations. (C) 2013 Elsevier B.V. All rights reserved.