Isomers of uroporphyrin free acids separated by HPLC

A synthetic mixture of uroporphyrin isomers I, II, III and IV as free acids in the synthetic ratio of 1:1:4:2 was resolved by reverse-phase HPLC using a C0:PEL (ODS) 37-50 micron precolumn and a Micro Bondapak C18 analytical column eluted with acetonitrile (4%) in phosphate buffer (pH 6.95). Clinically important I and III isomers of uroporphyrin were readily resolved directly from acidified urine as porphyrin free acids.