Preanalytical standardization of sphingosine-1-phosphate, sphinganine-1-phosphate and sphingosine analysis in human plasma by liquid chromatography-tandem mass spectrometry

Ceglarek U., Dittrich J., Helmschrodt C., Wagner K., Nofer J., Thiery J., Becker S.

Zusammenfassung

Background: Preanalytical standardization is required for a reliable quantification of the signaling molecules sphingosine-1-phosphate (S1P), sphinganine-1-phosphate (SA1P) and sphingosine (SPH). Methods: Methanolic protein precipitation of 15. μL EDTA-plasma was applied prior to analysis. Sphingolipids were separated in 3. min by hydrophilic interaction liquid chromatography (HILIC, SeQuant™ ZIC®-HILIC column) followed by tandem mass spectrometry. Stability of analytes in whole blood and plasma was investigated. Sphingolipid concentrations were determined in human plasma (n=50) and mice deficient in sphingosine kinase 1 (SK1) and 2 (SK2) (n=5). Results: Storing EDTA whole blood >. 60. min after blood withdrawal at room temperature resulted in an increase in S1P and SPH concentrations of ≥. 25%. Significant changes in SPH levels of +. 37% were observed after 60. min of storage of EDTA plasma at room temperature. Repeated freeze-thaw cycles of EDTA plasma resulted in increased S1P and SPH levels. Concentrations in human EDTA plasma were between 55.5 and 145.2. ng/mL for S1P and between 8.9 and 35.3. ng/mL for SA1P. Concentrations of S1P were 36% lower and 96% higher in EDTA-plasma from SK1- and SK2-deficient mice, respectively, compared to the wild type. Conclusions: Preanalytical standardization is a precondition for the analysis of sphingolipids in human blood. © 2014 Elsevier B.V.