Evaluating HPLC assay robustness with experimental design

A twelve run screening factorial experimental design was used to study the instrumental robustness of an HPLC weight percent assay for a fermentation derived pneumocandin B-0. The factors varied were the instrumental settings of wavelength, injection volume, flow rate, mobile phase composition, column temperature, and column lot. The measured responses were the fundamental liquid chromatographic parameters of retention time (RT); capacity factor (k'); theoretical plates (N); tailing factor (T); and resolution (Rs). The effect of each factor on the responses was calculated and significance determined by analysis of variance (ANOVA).