Preanalytical standardization of sphingosine-1-phosphate, sphinganine-1-phosphate and sphingosine analysis in human plasma by liquid chromatography-tandem mass spectrometry.
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.
Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Liebigstrasse 27, 04103 Leipzig, Germany; LIFE-Leipzig Research Center for Civilization Diseases, University Leipzig, Philipp-Rosenthal-Strasse 27, 04103 Leipzig, Germany.
Uta
U
Ceglarek
Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Liebigstrasse 27, 04103 Leipzig, Germany; LIFE-Leipzig Research Center for Civilization Diseases, University Leipzig, Philipp-Rosenthal-Strasse 27, 04103 Leipzig, Germany.
Julia
J
Dittrich
Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Liebigstrasse 27, 04103 Leipzig, Germany; LIFE-Leipzig Research Center for Civilization Diseases, University Leipzig, Philipp-Rosenthal-Strasse 27, 04103 Leipzig, Germany.
Christin
C
Helmschrodt
Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Liebigstrasse 27, 04103 Leipzig, Germany; LIFE-Leipzig Research Center for Civilization Diseases, University Leipzig, Philipp-Rosenthal-Strasse 27, 04103 Leipzig, Germany.
Kristin
K
Wagner
Center of Laboratory Medicine, University Hospital Münster, Albert-Schweizer-Campus 1, 48129 Münster, Germany.
Jerzy-Roch
JR
Nofer
Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Liebigstrasse 27, 04103 Leipzig, Germany; LIFE-Leipzig Research Center for Civilization Diseases, University Leipzig, Philipp-Rosenthal-Strasse 27, 04103 Leipzig, Germany.
Joachim
J
Thiery
Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Liebigstrasse 27, 04103 Leipzig, Germany; LIFE-Leipzig Research Center for Civilization Diseases, University Leipzig, Philipp-Rosenthal-Strasse 27, 04103 Leipzig, Germany. Electronic address: susen.becker@medizin.uni-leipzig.de.
Susen
S
Becker
D000818:Animals; D001774:Blood Chemical Analysis / Q000592:standards*; D002853:Chromatography, Liquid; D005260:Female; D064368:Healthy Volunteers; D006801:Humans; D008246:Lysophospholipids / Q000097:blood*; D008297:Male; D051379:Mice; D008875:Middle Aged; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000172:deficiency; D012015:Reference Standards; D015203:Reproducibility of Results; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000097:blood; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D000099157:Sphingosine Kinase
D008246:Lysophospholipids; D017853:Phosphotransferases (Alcohol Group Acceptor); D013110:Sphingosine; D000099157:Sphingosine Kinase; C060504:dihydrosphingosine 1-phosphate; C060506:sphingosine 1-phosphate
HILIC; Human plasma; Preanalytical standardization; Sphingolipids; Tandem mass spectrometry