High-pressure liquid chromatography of plasma free acid porphyrins

The separation and quantitation of plasma free acid porphyrins by high-pressure liquid chromatography and fluorescence is described. Porphyrins were extracted from plasma in a simple manner with a recovery >90%. They were separated by high-pressure liquid chromatography on a silica gel (10 μm) column, using a gradient of acetone:dilute acetic acid. Resolution of seven free acid porphyrin standards including coproporphyrins I and III, but not uroporphyrins I and III, was achieved in 12 min at picomolar concentrations. Plasma of patients with erythropoietic protoporphyria displayed protoporphyrin. Uroporphyrin was the only porphyrin found in plasma of eight patients with porphyria cutanea tarda. Normal plasma contained small amounts of uroporphyrin and/or traces of protoporphyrin.