Single step method for the accurate concentration determination of polysorbate 80

A simple high-performance liquid chromatography (HPLC) method is described that has the potential of determining the concentration of numerous classes of surfactants in the presence of protein and excipients. The method consists of a size-exclusion column and mobile phase containing a surfactant at concentrations above the critical micelle concentration for that surfactant. The presence of micellar surfactant in the mobile phase shortens the elution time of the surfactant resolving it from low-molecular-mass excipients and, in many cases, proteins (M-r<50 000). Polysorbate 80 standards show linearity from 2 to 1000 mu g/ml and have a coefficient of variation of less than 10% down to 7 mu g/ml. This method was demonstrated to be useful in the presence of human growth hormone (M-r 22 000) but showed accuracy down to only 50 mu g/ml. Use of an in-line protein A column (capable of binding IgG antibodies) with the size-exclusion column was shown to give similar detection limits in the presence of an intact antibody. (C) 1997 Elsevier Science B.V.