A method for separating ALA from ALA derivatives using ionic exchange extraction

Photodynamic therapy using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX is a recent approach to detect and treat some malignancies. The use of lipophilic derivatives of ALA has been exploited in the last years to enhance ALA penetration. In this paper, we describe the application of the Mauzerall and Granick’s method [J. Biol. Chem. 219 (1956) 435] to the quantification of ALA derivatives. We also describe the employment of reusable ion-exchange chromatographic columns for separating mixtures of ALA and ALA derivatives present in biological samples. The relation between 555 nm absorbance and ALA or ALA derivative concentration was linear up to 100 nmol/ml and the limit of detection of ALA and ALA derivatives was 1 nmol per ml. We employed a Dowex 50 X8 hydrogen form resin to separate ALA from the derivatives. Whereas 90 (plus-minus) 4% of the total ALA was eluted using sodium acetate, only 3–9% of the ALA derivatives was recovered. Only upon exposure of the resin to a high HCl concentration, the ALA derivatives were completely released. We employed this new method for the separation of ALA from ALA derivatives in cells exposed to different ALA compounds.