Reversed-phase high-performance liquid chromatography of salmon calcitonin and its degradation products in biological samples using column switching and flow-through radioisotope detection

For the determination of salmon calcitonin and its degradation products in biological samples, a reversed-phase HPLC method with column switching and flow-through radioisotope detection has been developed using high specific activity [¹²⁵I]salmon calcitonin. Effects of the precolumn packing material and washing solvent were examined in terms of [¹²⁵I]salmon calcitonin recovery. Spiked samples of [¹²⁵I]salmon calcitonin in plasma and kidney homogenate were injected onto a LiChroprep RP-8 precolumn after dilution with 0.1% trifluoroacetic acid. After washing the polar interfering compounds with 0.1% trifluoroacetic acid, the concentrated [¹²⁵I]salmon calcitonin and its degradation products were eluted and separated on a W-Porex C₁₈ column with a gradient of 0.1% trifluoroacetic acid in acetonitrile-water. Detection and calibration of [¹²⁵I]salmon calcitonin were possible down to picogram levels. Reproducible kinetic data for the degradation of intact [¹²⁵I]salmon calcitonin by rat kidney homogenate could be traced.