Postcolumn fluorescence as an alternative to evaporative light scattering detection for ceramide analysis with gradient elution in non-aqueous reversed-phase liquid chromatography.
Ceramide analysis was developed with gradient elution in non-aqueous reversed-phase liquid chromatography with evaporative light scattering detection (ELSD) or postcolumn fluorescence detection. Fluorescence detection (excitation, 360 nm; emission, 425 nm) after postcolumn formation of mixed assemblies between eluted ceramides and 1,6-diphenyl-1,3,5-hexatriene was developed. In comparison with ELSD, fluorescence detection allows a better detection of the minor species ceramide from ceramide type III (commercial mixture of non-hydroxy fatty acid-sphingosine) and appears to be more sensitive for quantitation of ceramides at low concentrations. The fluorescence response is linear over a wide range of injected amount of ceramide III (expressed as stearoyl-phytosphingosine): 10 ng to 1000 ng. The response of ELSD is non linear but can be linearized in double logarithmic coordinates for calculations over a narrow range, e.g. between 10 to 350 ng ceramide III injected. The lower quantitation limits of these two detectors are similar: 5 ng ceramide III was injected.
Zhou
J Y
JY
Laboratoire de Chimie Analytique, Faculté de Pharmacie, Chatenay-Malabry, France.
Chaminade
P
P
Gaudin
K
K
Prognon
P
P
Baillet
A
A
Ferrier
D
D
D002518:Ceramides / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008027:Light; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence
D002518:Ceramides