Fluorometry of singlet oxygen generated via a photosensitized reaction using folic acid and methotrexate

A method for the fluorometry of singlet oxygen (O-1(2)) using less fluorescent folic acid and its analogue, methotrexate (MTX), was examined. Folic acid and MTX were decomposed into a strongly fluorescent pteridine compound via a photosensitized reaction by O-1(2)- generating photosensitizers in a deuterium oxide solution. The fluorescence intensity increased in proportion to the irradiation time or the number of photons absorbed by the photosensitizer. This method using the fluorescence enhancement of these folic acid analogues can be applied to determine the quantum yield of O-1(2) generated through a photosensitized reaction in deuterium oxide. The background fluorescence of MTX is quite smaller than that of folic acid, indicating that MTX can be used for the more sensitive detection of O-1(2).