Journal of Chromatography B 2015-04-01

HPLC separation and ultrasensitive optical quantification of ceramide species applying 7-(diethylamino)coumarin-3-carbonyl azide derivatisation.

Markus Blaess, Ralf A Claus, Hans-Peter Deigner

Index: J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 986-987 , 123-8, (2015)

Full Text: HTML

Abstract

Ceramides are derivatised using 7-(diethylamino)coumarin-3-carbonyl azide; subsequent gradient HPLC separation allows sensitive optical quantification of individual cellular ceramides. Compared to 9-anthracenecarbonyl cyanide (9-anthroyl nitrile) as derivatisation agent, the limit of detection could be improved 415-fold, respectively 10,000-fold (detection limit 0.6 pmol labelled ceramide/sample) when compared to benzoyl chloride-labelling. Acidic or alkaline catalysts are not required, enabling drying and storing of the labelled samples and a free choice of solvents for subsequent HPLC-separation. The quantitative method is characterised by high sensitivity, linearity and robustness in the pico- to nanomolar concentration range and does not require mass-spectrometry for quantification of cellular ceramides.Copyright © 2015 Elsevier B.V. All rights reserved.