Selective and sensitive liquid chromatographic determination method of 5-hydroxyindoles with fluorous and fluorogenic derivatization.

Abstract A liquid chromatographic (LC) method with improved selectivity for the simultaneous determination of 5-hydroxyindoles (5-HIs; 5-hydroxytryptophan, 5-hydroxytryptamine, N -acetyl-5-hydroxytryptamine, 5-hydroxyindole-3-acetic acid, and 5-hydroxytryptophol) is described. This method involves precolumn derivatization with 4-(3′,3′,4′,4′,5′,5′,6′,6′,7′,7′,8′,8′,9′,9′,10′,10′,10′-heptadecafluorodecyl)benzylamine (HFBA) and separation of the derivatives using a fluorous LC column. In this study, stable benzoxazole derivatives of 5-HIs with HFBA have been obtained by a simple derivatization procedure; their fluorescent properties enabled highly sensitive detection. In addition, only the HFBA derivatives of 5-HIs has been selectively retained on the fluorous LC column via fluorous interaction whereby perfluoroalkyl compounds show affinities with each other, while the non-fluorous compounds did not. The HFBA derivatives were separated within 30 min and the detection limits for 5-HIs in a 20-μL injection volume were 1.2–14 fmol ( S/N  = 3). Furthermore, this method was applied to the analysis of 5-HIs in the human plasma from healthy subjects.