AN IMPROVED METHOD FOR THE DETERMINATION OF FREE SPHINGOSINE IN SERUM

A rapid, sensitive and selective high-performance liquid chromatographic method has been developed for the determination of sphingosine in human serum. After precipitation with methanol, the samples were extracted using Carbopack B disposable columns; the sphingosine was eluted with 0.05 M hydrochloric acid in methanol - dichloromethane (20: 80, v/v) and the extract evaporated to dryness at 40-degrees-C. The sample residue was then reconstituted with methanol and reacted with o-phthaldialdehyde reagent to produce a fluorescent compound. Separation was performed using an LC-18 column with 0.05 M phosphate buffer (pH 7) - methanol - acetonitrile (15 : 80: 5, v/v) as mobile phase. Fluorescence detection was performed with excitation and emission wavelengths of 340 and 455 nm, respectively. The serum extract was re-analyzed with a cyano LC columns to minimize the possibility of false positive results. The possible interference of compounds having a structure similar to that of sphingosine was evaluated. The mean recovery of sphingosine was > 94.5 %. The limit of detection of the assay was 1 ng mL-1. The between-run and within-run coefficients of variation for replicate analyses were < 4.0 % and < 3.4 %, respectively. The levels of free sphingosine in the serum of 40 normal subjects (20 male and 20 female) was investigated; the average level was 81.6 +/- 41.1 ng mL-1 (mean +/- S.D.) for males and 85.5 +/- 33.7 ng mL-1 for females.