Improved fluorescent determination method of cellular sphingoid bases in high-performanceliquid chromatography

Precolumn orthophthaldehyde (OPA) labeling method of sphingoid bases, sphingosine and sphinganine, was investigated to obtain high fluorescent detectability. In order to improve the fluorescent yield, we investigated the optimal solubility of sphingoid bases for five pre-incubation solvents by incorporating the heating procedure before OPA derivatization. The pre-incubation in ethanol prominently increased the fluorescent peak height of OPA derivative for each sphingoid bases in high performance liquid chromatography. About ten-fold increase of detectability was archived by pre-incubating lipid extracts pellets in ethanol at 60°C for 30 min. Optimal derivatization was performed in 30 min at ambient temperature and the fluorescent intensity of OPA derivative was stable for two weeks at 4° C. The detection limit of sphingosine was 0.1 pmol as injected amount. This method was applied to the determination of cellular sphingosine and sphinganine in various human lung cancer cells. This OPA procedure was prospective to be useful for quantitating the amount of sphingoid bases in other cancer cells.ope