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

Abstract

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 15μL EDTA-plasma was applied prior to analysis. Sphingolipids were separated in 3min 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 >60min 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 60min 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 145.2ng/mL for S1P and between 8.9 and 35.3ng/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.

Keywords: HILIC; Human plasma; Preanalytical standardization; Sphingolipids; Tandem mass spectrometry.