Determination of Human Epidermal Growth Factors in Cultured Media of Escherichia coli by High Performance Liquid Chromatography

Abstract

An automated HPLC method was proposed for the determination of a human epidermal growth factor, hEGF[1-53], and its fragments, hEGF[1-51] and hEGF[1-47], in the cultured medium of E, coli. The cultured medium was directly injected into the HPLC system which was composed of a short protein-coated octadecylsilane (ODS) precolumn for deproteinization and trapping of hEGFs and a usual ODS analytical column. By a column-switching technique, hEGFs trapped in the precolumn were transferred to the analytical column to allow their separation. The detection limits of hEGFs were ca. 100ng by UV detection at 210nm and were ca. 10ng by native fluorescene detection (Ex 287nm, Em 340nm). The recovery of the spiked hEGFs in the present method was almost quantitative (99.5-101.4%) with good reproducibility (less than 3% of RSD, within each run). The present method is simple and reproducible enough for the routine analysis of cultured medium for hEGFs.