Determination of Epidermal Growth Factors in Human Urine by High Performance Liquid Chromatography Using Anti-hEGF Antibody Precolumn

Abstract

A high performance liquid chromatographic method for the determination of human epidermal growth factors (hEGFs) in human urine is described. Using an anti-hEGF antibody column as a precolumn and an ODS column as an analytical column, each urine sample was directly injected onto the precolumn with aqueous mobile phase. With 0.2% trichloroacetic acid solution, the trapped hEGFs were transferred to the analytical column to allow their separation by reversed phase mode. The present method revealed that human urine contains hEGF[1-50], [1-51] and [1-52] in addition to hEGF[1-53]; the content determined by native fluorescence detection coincided with that by enzyme immunoassay. In the present method, the relative standard deviation (RSD) was less than 3% (n=6) for 25ng/ml of hEGF[1-53] and the detection limit was ca. 3ng for each hEGF, and the loading capacity of hEGF[1-53] on the anti-hEGF antibody precolumn was unchanged during analyses of 500 human urine samples.