Photodynamic therapy (PDT) is a new rapidly developing method for cancer treatment, which is based on the systemic administration of a tumor-localizing photosensitizer (PS), followed by irradiation of the tumor with visible spectrally adapted light.1-3 The photochemical interaction of the PS, light and molecular oxygen results in the formation of singlet oxygen ( 1 O 2 ) and other reactive oxygen species (ROS), which are believed to be toxic species responsible for induction of tumor necrosis.2,3 5,10,15,20-m-(Tetrahydroxyphenyl)chlorin (m-THPC), one of the second generation photosensitizers, 4 has recently been proposed as a new sensitizer for future use in the PDT of cancer.5,6 m-THPC is a single compound, derived from 5,10,15,20-meso-(tetrahydroxyphenyl)porphyrin (m-THPP), 4 with a maximum absorption in the red region of visible light (! max 652 nm). The latter property favors deeper light penetration into tissue and allows the method to achieve 6-10 mm depth of necrosis in tumor-bearing mice. As is the case for the majority of photosensitive drugs, m-THPC is degraded upon light illumination. This process, usually called 'photobleaching', involves a decrease in absorbance for certain spectral bands, variations of the fluorescence intensity or the formation of new absorption and emission bands.7 The m-THPC photodegradation has been observed in aqueous 8 and organic solutions, 9 in vitro, 10 in vivo in tumor-bearing mice 11 and in clinical practice.
12Studyies of the photobleaching reaction are particularly important for two reasons: (1) Identification of photobleaching products, which can be photodynamically active, thus opening the possibility for 'extended' photodynamic therapy; (2) Measurement of the photochemical reactions that reflect the potential therapeutic effect and thus help to match the light dose. This paper describes the first results concerning the application of matrix-assisted laser desorption/ionization coupled to Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICRMS) for the analysis of the parent m-THPC molecule and the products of photochemical m-THPC oxidation. We have studied the formation of photoproducts in organic solvents as a result of two consecutive irradiations separated by a dark storage period. The kinetics of photoproduct formation, assessed by MALDI, have been compared with that obtained by spectroscopic measurements.
EXPERIMENTALReagents and standard solutions m-THPC (Fig. 1) was kindly provided by Scotia Pharmaceuticals Ltd. (Guildford, UK) as crystalline powder (specification: content of m-THPC: 98.4% w/w; water content: 0.20% w/w; residual solvents: ethanol 0.18% w/w; content of impurities: m-THPP 0.7% w/w). Unless otherwise indicated, the photosensitizer was dissolved in methanol or in ethanol (Prolabo SA, Fontenay sous bois, France), which had been distilled beforehand twice under an inert atmosphere.
IlluminationIllumination was performed by exposing 3.5 mL of 7 mg/mL (10 À5 mol/L) of m-THPC in ethanol or methanol in a quartz cuvette (pathlength 1 c...
