Fluorescence and mass spectrometry studies of meta-tetra(hydroxyphenyl)chlorin photoproducts

The meta-tetra(hydroxyphenyl)chlorin (m-THPC), a second-generation sensitiz er used in photodynamic therapy (PDT), is currently under clinical trial. I n vivo fluorometry provides direct evidence that photobleaching processes a re induced at the tumor site during PDT, Photoproduct formation has thus to be taken into account to fully understand PDT treatment. A preliminary ste p is to determine the fluorescence characteristics of photoproducts formed in solution. Solutions of m-THPC irradiated at 514 nm have been separated b y HPLC using absorption and fluorescence detection. Six main photoproducts have been isolated. According to matrix-assisted laser desorption ionizatio n time-of-flight mass spectrometry (MALDI-TOFMS) results, five fluorescent photoproducts emitting at 652 ma have been attributed to three mono-, one d i- and one tri-hydroxy derivatives (m/z 697, 713 and 729, respectively), Fl uorescence characteristics of mono-hydroxy forms were found to be similar t o those of m-THPC, whereas fluorescence yields in di- and tri-hydroxy deriv atives were very low. Another product, corresponding to a MALDI-TOF MS main signal at mit 542, showed an absorption spectrum maximum at 322 nm while a weak fluorescence was detected at 480 nm, The loss of the Sorer band sugge sts that this photoproduct results from the opening of the reduced pyrrole ring. The part played by each of these products in the photobleaching pheno menon of m-THPC is discussed.