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

摘要

Background: Preanalytical standardization is required for a reliable quantification of the signaling molecules sphingosine-1-phosphate (SIP), sphinganine-1-phosphate (SA1P) and sphingosine (SPH). Methods: Methanolic protein precipitation of 15 uL EDTA-plasma was applied prior to analysis. Sphingolipids were separated in 3 min by hydrophilic interaction liquid chromatography (HILIC, SeQuant? ZIC?-HIL1C 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 (SKI) and 2 (SK2) (n = 5). Results: Storing EDTA whole blood >60 min after blood withdrawal at room temperature resulted in an increase in SIP 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 SIP and SPH levels. Concentrations in human EDTA plasma were between 55.5 and 145.2 ng/mL for SIP and between 8.9 and 35.3 ng/mL for SA1P. Concentrations of SIP were 36% lower and 96% higher in EDTA-plasma from SKI- and SK2-deficient mice, respectively, compared to the wild type.Conclusions: Preanalytical standardization is a precondition for the analysis of sphingolipids in human blood.