Quantitation of free sphingosine in liver by high-performance liquid chromatography

✍ Scribed by Alfred H. Merrill Jr.; Elaine Wang; Richard E. Mullins; W.Charles L. Jamison; Sanjay Nimkar; Dennis C. Liotta

• Publisher: Elsevier Science
• Year: 1988
• Tongue: English
• Weight: 738 KB
• Volume: 171
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(88)90500-3

✦ Synopsis

Conditions were established for the extraction of free sphingosine from liver and the separation and quantitation of this and other long-chain (sphingoid) bases (e.g., sphingosine, sphinganine, phytosphingosine, and homologs) by reverse-phase high-performance liquid chromatography (HPLC). The long-chain bases were extracted with chloroform and methanol and then treated with base to remove interfering lipids. After preparation of the o-phthalaldehyde derivatives, the long-chain bases could be separated using C18 columns eluted isocratically with methanol:5 mM potassium phosphate, pH 7.0 (90:10). The HPLC analyses took 15 to 20 min per sample and had lower limits of detection in the picomole range. Quantitation was facilitated by using a 20-carbon long-chain base homolog as an internal standard. The utility of the method was demonstrated with rat liver, providing the first quantitation of free sphingosine in this tissue of approximately 7 nmol/g wet wt.