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 revers ed-phase liquid chromatography with evaporative light scattering detection (ELSD) or postcolumn fluorescence detection. Fluorescence detection (excita tion, 360 nm; emission, 425 nm) after postcolumn formation of mixed assembl ies between eluted ceramides and 1,6-diphenyl-1,3,5-hexatriene was develope d. In comparison with ELSD, fluorescence detection, allows a better detecti on of the minor species ceramide from ceramide type III (commercial mixture of non-hydroxy fatty acid-sphingosine) and appears to be more sensitive fo r quantitation of ceramides at low concentrations. The fluorescence respons e 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 calc ulations over a narrow range, e.g. between 10 to 350 ng ceramide In injecte d. The lower quantitation limits of these two detectors are similar: 5 ng c eramide In was injected. (C) 1999 Elsevier Science BN. All rights reserved.