Precolumn orthophthaldehyde (OPA) labeling method of sphingoid bases, sphin
gosine and sphinganine, was investigated to obtain high fluorescent detecta
bility. In order to improve the fluorescent yield, we investigated the opti
mal solubility of sphingoid bases for five preincubation solvents by incorp
orating the heating procedure before OPA derivatization. The pre-incubation
in ethanol prominently increased the fluorescent peak height of OPA deriva
tive for each sphingoid bases in high performance liquid chromatography. Ab
out tenfold increase of detectability was archived by pre-incubating lipid
extracts pellets in ethanol at 60 degrees C for 30 min. Optimal derivatizat
ion was performed in 30 min at ambient temperature and the fluorescent inte
nsity of OPA derivative was stable for two weeks at 4 degrees C. The detect
ion limit of sphingosine was 0.1 pmol as injected amount This method was ap
plied to the determination of cellular sphingosine and sphinganine in vario
us human amount of sphingoid bases in other cancer cells.