Importance of the ionization states of m-THPC for its HPLC fluorescence detection

An original, rapid and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the detection of 5, 10, 15, 20 tetra-meso-hydroxyphenylchlorine (m-THPC), a photosensitizer used in the photodynamic therapy (PDT) of cancers. Chromatographic separation was carried out on a C(8) Zorbax column (80 x 4 mm, 5 microm). The mobile phase was an ethanol/aqueous sulphuric acid, pH 2.0 (65/35 v/v), in an isocratic mode yielding to rapid analysis (3.1 min) with narrow peaks. As the fluorescence intensity was found to be highly pH-dependent and to increase with pH values, a post-column device prior to the fluorescence detection (lambda(exc) = 423 nm, lambda(em) = 650 nm) was used to allow the addition of a 0.05 mol/L Na(2)HPO(4) solution to the mobile phase. Compared to standard conditions, a 300% increase of the fluorescence intensity was obtained for optimized operating conditions using experimental design. The validation of this analytical method showed that the response function was linear for concentrations up to 1000 microg/L (1.47 x 10(-6) mol/L) with a detection limit of 188 pg (S/N = 3).