Determination of 5-aminolaevulinic acid and porphobilinogen by high-performance liquid chromatography

A strong anion-exchange column coupled to a strong cation-exchange column with acetate buffer as eluent or reversed-phase ion-pair chromatography on octadecylsilica with methanol-water containing 1-heptanesulphonic acid as the mobile phase is described for the simultaneous separation of 5-aminolaevulinic acid and porphobilinogen. The separation is applied to the development of a fast and simple method for determining the activity of the enzyme aminolaevulinic acid dehydrase in human erythrocytes. 5-Aminolaevulinic acid is used as the enzyme substrate and the enzyme activity is expressed as micromoles of porphobilinogen formed per ml of erythrocytes in 1 h at 38 degrees. 5-Aminolaevulinic acid and porphobilinogen can also be separated from the urine of prophyric patients but the UV detector has insufficient sensitivity for the determination of 5-aminolaevulinic acid.