Analysis of commercial ceramides by non-aqueous reversed-phase liquid chromatography with evaporative light-scattering detection

Abstract

SummaryThis paper describes the development of a chromatographic system for analysis of commercial ceramides structurally similar to those found in the stratum corneum. The ceramides used in this study contain different amine bases (phytosphingosine, sphingosine and dihydrosphingosine) and fatty acids of different chain lengths and with different functional groups (hydroxylated and unsaturated). Non-aqueous reversed-phase (NARP) liquid chromatography with evaporative light-scattering detection (ELSD) were the techniques chosen in accordance with the nature of the ceramides. The eluent strength and the potential selectivity of different organic solvents were investigated. On a C18-bonded silica, the most promising chromatographic conditions employed a gradient from ACN-THF, 95:5, to ACN-THF-PrOH, 35:5:60, in 15 rain with a constant concentration of TEA (10 mM) and a stoichiometric amount of formic acid.

Wheat digalactosyldiacylglycerol molecular species profiling using porous graphitic carbon stationary phase

“…According to this scale, various strong solvents were selected: toluene, chloroform, dichloromethane, and tetrahydrofuran (THF). 0.1% of TEA and HCOOH in stoichiometric proportions was added to all solvents, in order to improve the ELSD response as previously described for the non-aqueous HPLC analysis of ceramides with ODS [22] and PGC phases [23]. Each pair of solvents was assessed with a binary linear gradient at a slope of 2% min -1 .…”

Response enhancement for the evaporative light scattering detection for the analysis of lipid classes and molecular species

“…Our assumption of a response detector phenomenon was assessed with FAMEs and wax by testing for two different types of elution modes: isocratic and gradient elution. The peak areas increased with the percentage of TEA/HCOOH until a plateau was reached, which greatly occured at a value of 0.1% [6]. Figure 1 presents the results of the addition of 0.1% (v/v) of TEA and an equimolar amount of HCOOH in the mobile phase.…”

“…The result of this chromatographic modification leads to an apparent improvement in detection. However, this resulted in a very slight decrease in retention and an important increase in the peak areas [6]. The use of another stationary phase porous graphitic carbon (PGC) which is particularly interesting for ceramide analysis [7], allowed us to partially verify the assumption of an effect on the detector response rather than an improvement of the column efficiency.…”

Use of principal component analysis for investigation of factors affecting retention behaviour of ceramides on porous graphitized carbon column

“…The chromatographic system on PGC was carried out in reversed-phase mode with a non-aqueous mobile phase which was in accordance with the highly hydrophobic nature of ceramides [7]. Solvent selection depends on the type of compound to be chromatographied as solvent strength in PGC is dependent on solute type [8].…”

“…These two solvents are generally found to be of low eluotropic strength on this reversed phase [8]. Stronger solvents were selected in accordance with their miscibility with methanol and acetonitrile and with the solvents already used for ceramide elution on C 18 silica in a previous study [7]. Moreover the strong solvents were chosen in various groups of selectivity.…”