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

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.