Reversed-phase liquid chromatography of radiolabeled peptides using a C18 guard-PAK precolumn system

In order to avoid radioactive contamination of high-performance liquid chromatography columns and injectors, we have investigated the use of a Guard-PAK precolumn system for the chromatography of [125I] labeled peptides. Two gonadotropin-releasing hormone analogs: 1) [D-Ala6-des-Gly10]-GnRH (GnRH-[Ala6]) and 2) [D-Ser(TBu)6-des-Gly10]-GnRH (GnRH-[Ser6]) and rat prolactin (r-PRL) were radiolabeled with 125I and subjected to reversed-phase liquid chromatography using a C18 Guard-PAK precolumn system. Major peak fractions of purified [125I]GnRH-[Ala6], [125I]GnRH-[Ser6], and [125I]r-PRL eluted at 24%, 28%, and 55% acetonitrile, respectively. Purified [125I]GnRH analogs showed specific high affinity binding to rat anterior pituitary gland membranes (specific activity: 1500-1700 Ci/mmol). Purified [125I]r-PRL showed high affinity binding to r-PRL antibody by RIA (specific activity: 70-75 microCi/micrograms). This rapid and efficient chromatographic method should be useful in the separation of a wide range of radiolabeled protein and peptide molecules.