High-performance liquid chromatographic analysis of biological and chemical heme polymerization

Free hematin can be converted to a stable polymer both chemically, by heating hematin in acid suspensions, or biologically, in the food vacuoles of malaria. A high-performance liquid chromatographic assay has been developed which can separate and quantitate both free hematin and the polymer (beta-hematin), based on the differential solubility of the two compounds. Ion-pair reverse-phase chromatography, utilizing tetramethylammonium chloride and heptane sulfonate as the ion-pair agents in the presence of 40% acetonitrile, was performed on a polymeric-resin-based column with a phenyl bonded phase. Initiating the runs at pH 2.5 led to elution only of the free hematin, and a subsequent shift to pH 12.0 converted the beta-hematin back to hematin which then eluted separately. The method was found to have a linear range of detection from 78 pmol to 20 nmol injected hematin and intra- and interday variations of 9.71 and 12.46%, respectively. The assay was used to study several basic aspects of heme polymerization in vitro, including effects of hematin and beta-hematin concentration on the rate of polymerization.