HPLC separation and ultrasensitive optical quantification of ceramide species applying 7-(diethylamino)coumarin-3-carbonyl azide derivatisation.
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.
Center for Sepsis Control and Care (CSCC), Jena University Hospital, Erlanger Allee 101, D-07747 Jena, Germany; Clinic for Anaesthesiology and Intensive Care, Jena University Hospital, Erlanger Allee 101, D-07747 Jena, Germany.
Markus
M
Blaess
Center for Sepsis Control and Care (CSCC), Jena University Hospital, Erlanger Allee 101, D-07747 Jena, Germany; Clinic for Anaesthesiology and Intensive Care, Jena University Hospital, Erlanger Allee 101, D-07747 Jena, Germany.
Ralf A
RA
Claus
Furtwangen University, Medical and Life Sciences Faculty, Jakob-Kienzle-Str. 17, D-78054 Villingen-Schwenningen, Germany. Electronic address: hans-peter.deigner@hs-furtwangen.de.
Hans-Peter
HP
Deigner
D002478:Cells, Cultured; D002518:Ceramides / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods; D003374:Coumarins / Q000737:chemistry*; D006801:Humans; D006834:Hydrazines / Q000737:chemistry*; D007962:Leukocytes; D057230:Limit of Detection; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
D002518:Ceramides; D003374:Coumarins; D006834:Hydrazines; C075233:7-diethylaminocoumarin-3-carbohydrazide
Cellular ceramide quantification; Fluorescence labelling; Reversed phase HPLC