Fluorescence detection of cardenolides in reversed-phase high-performance liquid chromatography after post-column derivatization
Belsner, K.; Büchele, B.
Journal of Chromatography. B, Biomedical Applications 682(1): 95-107
ISSN/ISBN: 1572-6495 PMID: 8832430 DOI: 10.1016/0378-4347(96)00056-4
Methods for the fluorescence derivatization of cardiac glycosides with concentrated acids from TLC are adopted to HPLC for post-column derivatization. The column effluent is blended with concentrated acids in a knitted tube reactor, which enables derivatization with negligible increase in chromatographic peak width. The selectivity of the reaction is temperature-dependent and influenced by the respective acid. Reactivity increases from H-3PO-4 fwdarw CH-3SO-3H tbd H-2SO-4. The conversion of digoxigenin, digitoxigenin and their digitoxosides is accelerated by Cu(II) acetate or Co(II) nitrate in H-2SO-4. Combined with a new two-mode, single-column solid-phase sample preparation, cardiac glycoside levels of less than 100 pg/glycoside in 1 ml plasma are detectable.