Summary
A high-performance liquid-chromatographic, column-switching system for automated sample pre-treatment and determination of human epidermal growth factor and its degradation products (hEGFs) is described. The system consists of an immunoaffinity precolumn (4.0×10mm) packed with diol silica immobilized with antibody against hEGF and an analytical ODS column (4.6×250mm). Samples such as cultured media ofE. coli, human urine, milk, seminal fluid and saliva can be directly injected on the immunoaffinity precolumn and the analytes of interest are trapped by the immobilized anti-hEGF antibody. After washing this precolumn with aqueous solvents, the analytes are desorbed with an aqueous solution of low pH and transferred to the analytical column to allow their separation in reversed phase mode. The recoveries of hEGFs spiked in these biological fluids were over 98%. The detection limit was 1 ng for a 1ml sample injection. This method was applied for the determination of hEGF levels in cultured media ofE. coli and biological fluids. Degradation of hEGF in human serum and urine was also examined.
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Hayashi, T., Sakamoto, S., Wada, I. et al. HPLC analysis of human epidermal growth factor using immunoaffinity precolumn. II. Determination of hEGFs in biological fluids. Chromatographia 27, 574–580 (1989). https://doi.org/10.1007/BF02258981
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DOI: https://doi.org/10.1007/BF02258981