A colorimetric assay for estimation of polyethylene glycol and polyethylene glycolated protein using ammonium ferrothiocyanate.

A colorimetric method for quantitative assay of polyethylene glycol (PEG) described here is based on partitioning of a chromophore present in ammonium ferrothiocyanate reagent from an aqueous to a chloroform phase in the presence of PEG. The method is simple, reproducible, and can detect PEG in amounts as low as 5 microg. It gives a linear response over a range of 5-100 microg. The absence of any interference by proteins makes the assay equally suitable for the estimation of PEG in PEG-protein conjugates. The method was employed to monitor the separation profile of a mixture of free and PEG-5000 coupled to bovine serum albumin during purification through a gel filtration column. In this report we have also demonstrated for the first time an assay method which permits a critical evaluation of pharmacokinetic properties of any PEG-protein conjugate under in vivo conditions.