An improved method for the determination of free sphingosine in serum |
| |
Authors: | A Laganà A Marino G Fago C Terregino B Pardo-Martinez |
| |
Institution: | (1) Department of Chemistry, University “La Sapienza”, Piazzale Aldo Moro 5, 00185 Roma, Italy;(2) Hospital Central Sur Petroleos Mexicanos, Mexico, D.F. |
| |
Abstract: | Summary 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°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 column 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. |
| |
Keywords: | Column liquid chromatography Solid phase extraction Carbopack B Free sphingosine in human plasma |
本文献已被 SpringerLink 等数据库收录! |
|