Reversed-phase high-performance liquid chromatography of radioiodinated salmon calcitonins

Reversed-phase HPLC conditions for simultaneous separation of salmon calcitonin, mono- and di-radioiodinated salmon calcitonins and their tryptic digested fragments have been developed. Salmon calcitonin was radioiodinated with (NaI)-I-125 by the iodo-beads method. After solid-phase extraction from the reaction mixtures using C-18 Bond Elut cartridges, mono- and di-radioiodinated salmon calcitonins were separated from each other, as well as from unlabeled salmon calcitonin, on a Bondclone 10 C-18 column (300 x 7.8 mm I.D.) by isocratic elution with 0.1% trifluoroacetic acid in 34% aqueous acetonitrile. The characteristics of either iodinated peptides or unlabeled salmon calcitonin were evaluated on the basis of UV absorbance (215 and 280 nm), fluorescence (lambda(ex) = 282 nm, lambda(em) = 310 nm) and measurement of specific radioactivity by means of a flow-through radio-isotope detector. HPLC separation of a tryptic digest of iodinated salmon calcitonin fraction on a W-porex 5 C-18 300 Angstrom column (250 x 4.6 mm I.D.) and subsequent amino acid analysis, led to the conclusion that radioiodination took place at the Tyr residue and not at the His moiety.