Separation and characterisation of sphingoceramides by high‐performance liquid chromatography‐electrospray ionisation mass spectrometry

Abstract

We developed a simple and reliable analytical method for the quantification and the characterization of ceramides extracted from biological samples by high-performance liquid chromatography (HPLC) coupled to electrospray ionisation tandem mass spectrometry (ESI/MS/MS). The chromatographic separation of analytes was carried out in a RP8 column, eluting with a methanol-water mixture in gradient elution mode. The separated lipids were detected by total ion monitoring and characterised by MS/MS spectra; quantitative analysis was performed by integrating the extracted ion peaks obtained in the negative ion mode. Good repeatability was obtained for retention time (0.3-2%), peak area ratio (A(S)/A(IS), 2-8%), as well as limit of detection (LOD, 5-26 pg) and quantification (LOQ, 13-53 pg). The method was validated for the analysis of N-palmitoyl-D-erythro-sphingosine (Cer16), N-stearoyl-D-erythro-sphingosine (Cer18), N-tetracosanoyl-D-erythro-sphingosine (N24:0, lignoceric ceramide, Cer24:0), and N-tetracos-15'-enoyl-D-erythro-sphingosine (N24:1, nervonic ceramide, Cer24:1), giving good results. Lipid mixtures, extracted from skin and epidermal cells, were analysed for their content of the studied ceramides.

Comprehensive quantification of ceramide species in human stratum corneum

“…The mixed authentic solution at a concentration of 4 nM each (40 fmol in 10 l injection volume) was applied to the simultaneous SIM measurement in NPLC-ESI-MS, followed by the calculation of LOD and LOQ as signal to noise (S/N) being 3 and 10, respectively. Calibration curves were produced by injecting the mixed authentic solutions in a range between 4 and 128 nM (4,8,16,32,64, and 128 nM corresponding to 40, 80, 160, 320, 640, and 1280 fmol in 10 l injection volumes, respectively). The concentration range for the calibration curves was based on preliminary experiments of the dynamic ranges.…”

“…This indicates However, there seemed to be no tendency that specifi c fatty acid or sphingoid moieties are related to the greater RSD. This might be related to relatively unstable performances of NPLC-ESI-MS, especially on separate days, compared with RPLC-ESI-MS that is stable and commonly used in the analyses of CERs (22,(30)(31)(32). Relative response values are a ratio of the slope of the calibration curve (100-1000 fmol) each for 11 authentic CERs to that of the internal standard CER[N(17)S (18) (3) responses of isobaric species may be substantially similar to each other.…”

“…To characterize differences in lipidomes among biological samples of interest, the relative intensities or areas given from mass spectrometers are only helpful as semi-quantitative values ( 11,12 ). Precise quantitative values of lipidomes of interest would also be desirable, especially in the case of evaluations of lipid dynamics in de MS has been employed for the analysis of CERs in human SC as follows: reversed-phase LC (RPLC)-ESI-MS ( 22,(30)(31)(32); ESI-MS ( 33,34 ); RPLC-thermospray-MS ( 35 ); and NPLC-APCI-MS (36)(37)(38)(39). However, those methods were not validated as quantitative methods for overall CER species in the SC, although some studies reported quantitative values based on unrealistic assumptions.…”

Analysis of phospholipids in membrane of Acidithiobacillus ferrooxidans grown in liquid (ferrous sulphate and sodium thiosulphate) and solid (sulfur) substrate

“…These bilayer membranes provide shape for the cell and its organelles. It also helps cell to maintain its physiochemical properties, creating an environment inside a cell that is necessary for proteins to interact and function [2]. Therefore even a minute change in membrane phospholipids can lead to drastic change in the membrane causing major defect in cell functions and viability.…”

A review of chromatographic methods for the assessment of phospholipids in biological samples

“…Finally, lipids are precursors for numerous second messengers. For example, sphingolipids are enzymatically broken down into ceramides, which are important signaling molecules involved in the modulation of cell proliferation, cell cycle arrest and senescence (Camera et al, 2004). Phospholipids are precursors to arachidonic acid, which can be enzymatically broken down to leukotrienes, prostaglandins and thromboxanes, all important modulators of inflammation.…”