High-Performance Liquid Chromatographic Determination of Iron-Containing Proteins with On-Line Inductively Coupled Plasma Mass Spectrometric Detection

Abstract

An inductively coupled plasma mass spectrometer was employed as an iron detector in gel permeation liquid chromatography to separate ferritin, hemoglobin, myoglobin, and cytochrome-c, by use of potassium hexacyanoferrate(III) as the iron standard. The absolute detection limits for the four proteins were 0.01, 1, 0.7, and 0.4μg, respectively, at 10μl injections. These values were an order-of-magnitude lower than those obtained by using an inductively coupled plasma atomic emission spectrometer as detector. From the peak area in the Fe chromatograms, the number of Fe atoms per molecule was evaluated for myoglobin and cytochrome-c to be 0.97 and 1.0, respectively.