Separation of haem compounds by reversed-phase ion-pair high-performance liquid chromatography and its application in the assay of ferrochelatase activity.

The separation of haems and porphyrins was achieved in a reversed-phase ion-pair high-performance liquid chromatography system using tetrabutylammonium hydrogen sulphate as the pairing ion. The concentration of methanol and pH in the mobile phase were determinative parameters for the elution pattern of the compounds. Two isocratic systems--one for the assay of protohaem IX and one for deuterohaem IX--were developed. The chromatographic systems were applied to the assay of ferrochelatase activity in mitochondria using either protoporphyrin or deuteroporphyrin as the substrate. The ferrochelatase activity was also measured in reticulocytes, which contain high levels of endogenous haem.