Comparative study of naphthalene-2,3-dicarboxaldehyde and o-phthalaldehyde fluorogenic reagents for chromatographic detection of sphingoid bases

Naphthalene-2,3-dialdehyde (NDA) was developed as a precolumn labeling reagent for the fluorescent determination in a HPLC system of bioactive sphingoid bases, including sphingosine, sphinganine, and C20-sphinganine. Cellular sphingoid bases generally exist in the range of 10∼100 pmol/106 cells in a wide variety of cell types and tissues. This study aimed to obtain stable fluorescent derivatives of sphingoid bases and to increase their detectability at low concentrations. Sphingoid bases were reacted with NDA in the presence of cyanide ion to readily make an intensely fluorescent structure, 1-cyano-2-alkyl-benz[f]isoindole (CBI) and were then eluted separately on a reversed-phase C18 column with a simple mobile phase of 90% acetonitrile. For evaluating the NDA method, we compared the fluorescent intensity, elution profile, stability, and detectability of NDA derivatives with those of corresponding o-phthalaldehyde (OPA) derivatives. By monitoring the fluorescent intensity at the excitation wavelength of 252 nm and emission wavelength of 483 nm, NDA derivatives were sensitively determined at concentrations below 1.0 pmol of sphingoid bases in 1·105 U937 cells and were more stable than OPA derivatives. Linear calibration plots were obtained in the range studied (0.5∼500 nM). The limit of detection for NDA derivatives of sphingoid bases was approximately 0.1 pmol (S/N=3). The method successfully measured the accumulation of sphingosine in U937 cells following N,N-dimethylsphingosine treatment, and of sphinganine following fumonisin B1 treatment.