Determination of sphingosine and sphinganine in human urine by high performance liquid chromatography

An improved liquid chromatographic method for the determination of sphingosine(So) and sphinganine(Sa) in human urine was-developed. It involves isolation of exfoliated cells from human urine followed by a rapid and efficient extraction of sphingolipid bases in ethylacetate, and a derivatization step with o-phthaldialdehyde and a high performance liquid chromatographic separation on a 15 cm x 3.9 mm, 4 mu m Nova-Pak C-18-RP column, with a phosphate buffer/methanol gradient. Fluorescence was monitored at 335 nm for excitation and 440 nm for emission. The limits of detection were 0.05 ng or 0.075 mu g/L in female urine and 0.005 mu g/L in male urine. The method was applied to analyze 40 human urine samples collected from a village in China. The So, Sa and Sa/So ratio in female urine varied from 1.29 similar to 13.58 mu g/L, 0.25 similar to 3.13 mu g/L and 0.15 similar to 0.25, respectively, while those in male urine varied from 0.075 similar to 3.07 mu g/L, 0.019 similar to 0.50 mu g/L and 0.028 similar to 0.26, respectively.