Simultaneous Quantification of Sphingolipids in Small Quantities of Liver by LC-MS/MS

Abstract

Sph, S1P, and Cer, derived from the membrane sphingolipids, act as intracellular and intercellular mediators, involved in various (path) physiological functions. Accordingly, determining the distributions and concentrations of these sphingolipid mediators in body tissues is an important task. Consequently, a method for determination of sphingolipids in small quantities of tissue is required. Sphingolipids analysis has been dependent on improvements in mass spectrometry (MS) technology. Additionally, decomposition of sphingosine-1-phosphate (S1P) in the tissue samples before preparation for MS has hindered analysis. In the present study, a method for stabilization of liver samples before MS preparation was developed using a heat stabilizer (Stabilizor™ T1). Then, a LC-MS/MS method using a triple-quadrupole mass spectrometer with a C8 column was developed for simultaneous determination of sphingolipids in small quantities of liver specimens. This method showed good separation and validation results. Separation was performed with a gradient elution of solvent A (5 mmol L⁻¹ ammonium formate in water, pH 4.0) and solvent B (5 mmol L⁻¹ ammonium formate in 95% acetonitrile, pH 4.0) at 300 μL min⁻¹. The lower limit of quantification was less than 132 pmol L⁻¹, and this method was accurate (∼13.5%) and precise (∼7.13%) for S1P analysis. The method can be used to show the tissue distribution of sphingolipids.