Separation and Detection of Ceramides by HPLC Followed by Evaporative Light-Scattering Detection and Thin Layer Chromatography

Ceramides are important signaling molecules involved in a variety of cellular processes, including cell growth, differentiation, and apoptosis. Currently, different methods are used for ceramide analysis, some of which are insensitive or cumbersome. This paper described methods utilizing thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) followed by evaporative light scattering detection (ELSD) to detect ceramide directly in cell extracts without derivatization, which was proved to be efficient and reproducible. Five kinds of ceramides were used as standards. Both TLC and normal-phase HPLC/ELSD results indicate that yeast contains several kinds of ceramides.

Analysis of Hupehenine in the Total Alkaloids from Fritillaria hupehensis by HPLC-ELSD

A reverse-phase high pressure liquid chromatography （HPLC） method with evaporative light scattering detection （ELSD） has been developed for the quantitative analysis of hupehenine in the total alkaloids from Fritillaria hupehensis. Samples were analyzed on a reverse-phase column （Hypersil C-18） with a mobile phase of methanol：water：chloroform： triethylamine （85：15：1：0.6）. The ELSD was set at the drift tube temperature of 68.3℃ and gas flow rate of 1.8 L/min. Hupehenine＇s retention time was 13.7 min with an asymmetry factor of 1.2. The validity of the method has been verified with linearity, limit of detection, accuracy and precision. The logarithmic linear curve was obtained from 8.936 to 134.04 μg/mL （r=0.9993）. The detection limit （S/N〉3） of hupehenine was 1.79 μg/mL on the column. Intra-day RSD was 1.42% and inter-day RSD was 2.26% （3 days within a week）. The average recovery of hupehenine was 101.50%, and RSD was 1.62%.

Chemical comparison of dried rehmannia root and prepared rehmannia root by UPLC-TOF MS and HPLC-ELSD with multivariate statistical analysis

To identify the chemical differences which lead to the different therapeutic effects of dried rehmannia root(DRR)and prepared rehmannia root(PRR),we compared the chemical composition of decoctions of randomly purchased DRR and PRR using ultra performance liquid chromatography(UPLC)coupled with time-of-fight mass spectrometry and high performance liquid chromatography(HPLC)coupled with evaporative light scattering detection(ELSD)with the aid of multivariate statistical analysis.Both approaches clearly revealed compositional and quantitative differences between DRR and PRR.UPLC-MS data indicated stachyose,rehmaiono-side A(or rehmaionoside B),acteoside(or forsythiaside,or isoacteoside),6-O-coumaroylajugol(or 6-O-E-feruloylajugol,or 6-O-Z-feruloylajugol)as important discriminators between DRR and PRR decoctions.HPLC-ELSD analysis showed that the content of fructose in the decoctions of PRR was about four times greater than that of DRR(P<10^(-5)),while sucrose content in the decoctions of PRR was only about one seventh of that in DRR(P<0.01).Our results suggest that some compounds,such as fructose,stachyose and rehmaionoside,may be responsible for the differing therapeutic effects of DRR and PRR.Furthermore,improvements in quality control for PRR,which is currently lacking in the Chinese Pharmacopoeia,are recommended.