Simple sensitive and simultaneous high-performance liquid chromatography method of glucoconjugated and non-glucoconjugated porphyrins and chlorins using near infra-red fluorescence detection

This paper reports, for the first time, a reversed-phase high performance liquid chromatographic method for the simultaneous determination of seven glucoconjugated and non-glucoconjugated porphyrins and chlorins, using near infra-red fluorescence detection. Chromatographic separation was performed on nucleosil-CN analytical column using an isocratic acetonitrile-0.1% (w/v) TFA at pH 1.8 (55:45, v/v) as mobile phase. Wavelength gradient was employed for sensitive detection, porphyrins derivates were monitored at lambda(exc) = 440 nm and lambda(emi) = 680 nm; and chlorins derivates at lambda(exc) = 420 nm, lambda(emi) = 650 nm. The method was validated and applied to monitor the biodegradation of a tri glucoconjugated chlorin derivative, TPC(glu)3, in spiked samples of human serum.