ISSN:
1612-1112
Keywords:
Column liquid chromatography
;
Solid phase extraction
;
Carbopack B
;
Free sphingosine in human plasma
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF02320464
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