Simultaneous quantitative determination method for ceramide species from crude cellular extracts by high-performance liquid chromatography–thermospray mass spectrometry

I have developed a simple method which enabled simultaneous analysis of ceramides in the subcellular fractions from cultured cells by HPLC–thermospray mass spectrometry. The HPLC–thermospray mass spectra from ceramide standards were characterized by the high intensity of the MNa+ and MH+–H2O ions. As the other minor ions, MK+, MH+ and m/z 282 ions were detected. Although the preponderance of MNa+ ions compared with the MH+–H2O ions was detected in non-hydroxy fatty acid-ceramides, the preponderance of MH+–H2O ions based on the elimination of the hydroxyl group introduced at the α-position of acyl-portion compared with the MNa+ ions was detected in α-hydroxy fatty acid-ceramides. In calibrations for authentic ceramides using N-octanoylsphingosine as an internal standard, an approximately linear relationship existed between the ratios of peak-areas of each ceramide to that of the internal standard and the known amounts of each ceramide. The factor (f) of each ceramide was calculated as follows; N-oleoyl-d-sphingosine (f=0.45), N-palmitoyl-d-sphingosine (f=0.40), N-stearoyl-d-sphingosine (f=0.39), N-nervonoyl-d-sphingosine (f=0.39) and N-lignoceroyl-d-sphingosine (f=0.35). In subcellular fractions from A549 and HepG2 cells, although ceramide species content per mg protein was high in the nuclear envelope fractions, the 7000 g pellet fractions and the 100 000 g pellet fractions, a large portion of the ceramide species was concentrated in the nuclear envelope fraction. In addition, this method was applied to a mild alkaline hydrolyzate of total ceramides from pig stratum corneum, and MNa+/MH+–H2O ions corresponding to several ω-hydroxyacyl-ceramides were detected.