DETERMINATION OF PORPHYRINS IN HUMAN BLOOD BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A method for the reversed-phase high performance liquid chromatography (HPLC) of porphyrin carboxylic acids and zinc protoporphyrin with fluorescence detection is described. The method is applied to determine the porphyrins in human blood. Erythrocyte porphyrins were extracted from whole blood with 300 μl of acidic methanol solution. Metalloporphyrin was separately extracted with an acetone-pyridine-sterox solution from another aliquot of blood. The supernatants from both extracts were simultaneously injected onto a μBondapak C18 column for detection of porphyrins with excitation wavelength at 405 nm and emission wavelength at 630 nm. The separation required a pre-treatment of the column with a binary mobile phase containing 0.1 M sodium phosphate in acetonitrile (29 : 16, v/v, pH 5.3) for 10 min prior to sample injection. The separation was completed isocratically by increasing the acetonitrile concentration in the mobile phase (0.1 M sodium phosphate : acetonitrile = 18: 129, v/v, pH 5.3) 4 min after injection. The elution of all the compounds took less than 23 min. © 1990, Taylor & Francis Group, LLC. All rights reserved.