On the presence of phosphorylated sphingoid bases in rat tissues. A mass-spectrometric approach

A simple and straightforward procedure to analyze phosphorylated sphingoid bases has been developed. After phase separation of lipid extracts under alkaline conditions, the compounds were quantitatively recovered in the aqueous upper phase. Following a clean-up of the aqueous phase on C18-solid phase extraction columns, the amino-group of the bases was derivatized by means of phenylisothiocyanate addition. FAB-MS of the phenylthiocarbamate derivatives of sphingenine- and sphinganine-phosphate in the negative mode revealed the expected pseudo-molecular ions (M-1) at 513 m/z and 515 m/z, respectively. Moreover, a typical fragmentation pattern, characterized by the loss of the phenylthiocarbamate moiety (m/z = 135), was observed. When applied to rat tissues, the presence of sphingenine-phosphate in brain, kidney and liver could easily be demonstrated. Highest levels, amounting to 5 nmol/g of wet weight, were present in brain.