Isocratic ion-pair high-performance liquid chromatographic methods for the determination of uroporphyrin and coproporphyrin type II and IV isomers in human urine.

Urinary porphyrins of porphyric patients were isolated as their methyl esters by using a simple, modified thin-layer chromatographic system. Existing methods for the isocratic ion-pair high-performance liquid chromatographic separation of uroporphyrin and coproporphyrin isomers were decisively improved by elevating the column temperatures, changing the types of columns used and modifying the eluent compositions. These techniques were applied to the determination of the isomeric distribution of uroporphyrins and coproporphyrins isolated from urines of patients in the acute or latent phase of acute intermittent porphyria. In these urines relatively high contents of the atypical uroporphyrins II (2-5%) and IV (13-19%) were found. The coproporphyrin fractions contained significantly smaller amounts of the atypical isomers II (1-2%) and IV (2-5%), the presence of which was demonstrated for the first time in such urines. Several mechanisms for the formation of the atypical coproporphyrin isomers are discussed. The isocratic ion-pair separation method served also to control the isomeric purity of uroporphyrin specimens of both natural and synthetic origin.