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

An original, rapid and sensitive high-performance liquid chromatographic (H PLC) method has been developed for the detection of 5, 10, 15, 20 tetra-mes o-hydroxyphenylchlorine (m-THPC), a photosensitizer used in the photodynami c therapy (PDT) of cancers. Chromatographic separation was carried but on a C-8 Zorbax(R) column (80 x 4 mm, 5 mum). The mobile phase was an ethanol/a queous 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, l ambda (em) = 650 nm) was used to allow the addition of a 0.05 mol/L Na2HPO4 solution to the mobile phase. Compared to standard conditions, a 300% incr ease of the fluorescence intensity was obtained for optimized operating con ditions using experimental design. The validation of this analytical method showed;hat the response function was linear for concentrations up to 1000 mug/L (1.47 x 10(-6) mol/L) with a detection limit of 188 pg (S/N = 3). Cop yright (C) 2001 John Wiley & Sons, Ltd.