A comparative study of normal and reverse phase high pressure liquid chromatography for analysis of porphyrins accumulated after 5-aminolaevulinic acid treatment of colon adenocarcinoma cells

Primary adenocarcinoma cells of the rectosigmoid colon (WiDr-cells) were tr eated with 5-aminolevulinic acid (5-ALA). Cellular porphyrins were separate d and quantified by high performance liquid chromatography (HPLC), both as free porphyrin acids after an easy extraction method with a subsequent reve rse phase technique, and then as porphyrin esters after a more laborious ex traction method and subsequent normal phase technique. The porphyrins were detected by means of a fluorescence detector. Analysis by normal phase HPLC indicated that 81% (739 pmol/mg protein) of the total amounts of fluoresci ng porphyrins accumulated was protoporphyrin IX, while similar analysis by reverse phase HPLC indicated that PpIX constituted 91% (622 pmol/mg protein ) of the accumulated porphyrins. In addition to protoporhyrin IX, copro-, h exa-, hepta- and uroporphyrins were observed in extracts from 5-ALA-treated cells by both methods. The discrepancy between the two methods increased w ith increasing hydrophilicity of the analysed porphyrins, with uroporphyrin estimated to be 6-fold higher (63 vs. 10 pmol/mg protein) by normal than b y reverse phase HPLC. (C) 2000 Elsevier Science Ireland Ltd. All rights res erved.