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Sphingolipids are an important group of lipids that play crucial roles in living cells, facilitating cell recognition, signal transduction and endocytosis. The concentration of sphingosine and some of its derivatives like sphinganine may serve as a biomarker for the diagnosis of sphingolipidoses or be used for further research into similar diseases. In this study, a sphingolipid extraction and a high resolution detection method specific for sphingosine and sphinganine was adapted and tested. Lipids were extracted from rats' serum, coupled to o-phthalaldehyde and detected with a fluorescence detector after running through a silica gel column in a high performance liquid chromatography system. With this method, we analysed 20 male and 20 female rat serum samples and compared the concentrations of sphingosine and sphinganine. The results showed a significant difference between the sphingosine concentrations in the male and female rats. The sphingosine concentration in female rats was 805 ng/mL (standard deviation, SD ± 549), while that in males was significantly lower at (75 ng/mL (SD ± 40)). Furthermore, the sphingosine:sphinganine ratio was almost 15-fold higher in the females' samples. The method presented here facilitates the accurate quantification of sphingosine and sphinganine concentrations down to 2.6 ng and 3.0 ng, respectively, and their ratio in small amounts of rat serum samples to study the sphingolipid metabolism and its potential modulation due to gene mutations or the effect of prevalent toxins. [ABSTRACT FROM AUTHOR]