Quantification of urinary porphyrins by liquid chromatography after oxidation of porphyrinogens

A quantitative "high-performance" liquid-chromatographic method is described for determining porphyrins in human urine. Porphyrinogens in urine are first converted to the corresponding porphyrins by oxidation with iodine. Uroporphyrin, hepatacarboxylic acid porphyrin, hexacarboxylic acid porphyrin, pentacarboxylic acid porphyrin, and coproporphyrin I and III isomers are then separated on a reversed-phase column and measured by fluorometry. Analysis for the six porphyrins is complete within 24 min, including reconditioning for the next sample. The detection limit (twice the signal/noise ratio) for each porphyrin was 1 nmol/L for urine (25 fmol per 50-microL injection). Mean analytical recovery of each porphyrin ranged from 85% to 91%, within-day CVs from 1.4% to 7.3%. Normal reference intervals for porphyrins were established by assaying urine samples from 75 healthy subjects. Significant sex-related differences in coproporphyrin I and III isomers were evident when the values were expressed as nanomoles per gram of creatinine. Coproporphyrin isomer ratio was estimated for utility in the diagnosis of porphyrinurias.