Enhanced detection of seven glucoconjugated and hydroxylated porphyrins and chlorins by nonaqueous capillary electrophoresis combined with stacking

The nonaqueous capillary electrophoresis mode which includes a preconcentration step based on a transient pseudo-isotachophoresis to the simultaneous separation of seven glucoconjugated and hydroxylated porphyrins and chlorins, exhibiting very close structures, is reported. A high methanol content, of the buffer solution, was necessary in order to prevent self-assembly of the compounds and to enhance their solubility during separation. With the addition of 66% (v/v) methanol and 1% (w/v) NaCl in the aqueous sample solution, large volumes could be injected (44% capillary volume) without a loss in resolution. Sensitivity of detection was therefore improved by a 100-fold factor with regard to the method employing normal injection (2% capillary volume). Optimum electrophoretic conditions, in terms of sensitivity and performance, were obtained by using 20 mM phosphoric acid buffer, pH 2.2 and 50% methanol. The method was validated and applied to qualitative analysis of glucoconjugates in serum samples.