Considerations in the development of a sensitive HPLC assay for human epidermal growth factors in human plasma

A sensitive assay was developed for human epidermal growth factors (hEGF) 1–48 (dosed), hEGF 1–53 (endogenous), without interference from potential metabolites hEGFs 1–47 or 1–46. Spiked human plasma samples were injected directly, utilizing on-line immunoaffinity HPLC (anti-hEGF) clean-up. No change in capacity was noted after 81 cycles. After release from the immunoaffinity column, the fragments were further resolved by strong cation-exchange (SCX) via a column switching valve. Method development also required interfacing immunoaffinity, ion-exchange, and detection components. Immunoassays on collected fractions yielded a detection limit of 1 μg ml −1, although a detection limit of 75 pg ml −1 appears feasible.