High-Throughput Analysis of Sphingosine 1-Phosphate, Sphinganine 1-Phosphate, and Lysophosphatidic Acid in Plasma Samples by Liquid Chromatography–Tandem Mass Spectrometry

Abstract

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are ubiquitous lipid messengers found in the blood and most cell types. Both lysophospholipids are ligands of G protein-coupled receptors and mediate important physiological processes. Moreover, lysophospholipids are potential biomarkers for various diseases, including atherosclerosis and cancer. Because existing methodologies are of limited value for systematic evaluations of S1P and LPA in clinical studies, we developed a fast and simple quantification method that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS). Sphingoid base 1-phosphates and LPA species were quantified in negative-ion mode with fragments of m/z 79 and 153, respectively. The internal standards LPA 17:0 and [(13)C(2)D(2)]S1P were added before butanol extraction. Application of hydrophilic-interaction chromatography allowed coelution of analytes and internal standards with a short analysis time of 2.5 min. Comparison of butanol extraction with a frequently used extraction method based on strong acidification of human plasma revealed artificial formation of LPA from lysophosphatidylcholine with the latter method. Validation according to US Food and Drug Administration guidelines showed an overall imprecision (CV) of <12% and a limit of detection <6 nmol/L for all lysophospholipid species. Concentrations of S1P and sphinganine 1-phosphate (SA1P) in EDTA-containing plasma were stable for 24 h at room temperature, whereas LPA concentrations increased substantially over this period. Our validated LC-MS/MS methodology for quantifying LPA, S1P, and SA1P features simple sample preparation and short analysis times, therefore providing a valuable tool for diagnostic evaluation of these lysophospholipids as biomarkers.