Comprehensive Quantitative Analysis of Bioactive Sphingolipids by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

Abstract

There has been a recent explosion in research concerning novel bioactive sphingolipids (SPLs) such as ceramide (Cer), sphingosine (Sph), and sphingosine 1-phosphate (Sph-1P) and this has necessitated the development of accurate and user-friendly methodology for analyzing and quantitating the endogenous levels of these molecules. ESI/MS/MS methodology provides a universal tool used for detecting and monitoring changes in SPL levels and composition from biological materials. Simultaneous ESI/MS/MS analysis of sphingoid bases (SBs), sphingoid base 1-phosphates (SB-1Ps), ceramides (Cers), ceramide 1-phosphates (Cer-1P), glucosyl/galactosyl-ceramides (Glu-Cers), and sphingomyelins (SMs) is performed on a Thermo Fisher Scientific triple quadrupole mass spectrometer operating in a multiple reaction monitoring (MRM) positive ionization mode. Biological materials (cells, tissues, or physiological fluids) are fortified with internal standards (ISs), extracted into a one-phase neutral organic solvent system, and analyzed by a LC/MS/MS system. Qualitative analysis (identification) of SPLs is performed by a Parent Ion scan of a common fragment ion characteristic for a particular class of SPLs. Quantitative analysis is based on calibration curves generated by spiking an artificial matrix with known amounts of target analyte, synthetic standards, and an equal amount of IS. The calibration curves are constructed by plotting the peak area ratios of analyte to the respective IS against concentration, using a linear regression model. This robust analytical procedure can determine the composition of endogenous sphingolipids (ESPLs) in varied biological materials and achieve a detection limit of subpicomole level. This methodology constitutes a "Lipidomic" approach to study the SPLs metabolism, defining a function of distinct subspecies of individual bioactive SPL classes.

Cytokine-induced release of ceramide-enriched exosomes as a mediator of cell death signaling in an oligodendroglioma cell line

“…The pellets were lysed in RIPA buffer and an aliquot used for protein estimation by BCA method (38) and the remainder kept frozen at 80°C until used for sphingolipidomic analysis. A HPLC-MS/MS analysis of endogenous Cer, dihydroceramide (dhCer), and SM components was performed on a ThermoFisher TSQ Quantum or SCIEX Q-Trap triple-stage quadrupole mass spectrometer, operating in a multiple reaction monitoring positive ionization mode, as previously described (39,40). Briefly, test samples were fortified with the internal standards (ISs) , and extracted into a one-phase solvent system with ethyl acetate/ isopropanol/water (60/30/10%; v/v).…”

Renal Glycosphingolipid Metabolism Is Dysfunctional in Lupus Nephritis

“…Quantitation of sphingolipid species was performed by the Lipidomics Core at Medical University of South Carolina on a Thermo Finnigan TSQ 7000 Triple-Stage Quadrupole Mass Spectrometer operating in a Multiple Reaction Monitoring (MRM)-positive ionization mode as described. 37,38 ESI/MS/MS analyses of endogenous GlcCer and GalCer molecular species (C18-Sphingoid Base) were performed on a Thermo Fisher TSQ Quantum Triple Quadrupole Mass Spectrometer operating in an MRMpositive ionization mode using a modified version. 38 Briefly, cell pellets corresponding to about 1-3310 6 cells were fortified with synthetic and not natural internal standards (ISs: C8-GluCer, C8-GalCer, C12-GluCer, and C12-GalCer; Avanti Polar Lipids, Inc., Alabaster, AL) and extracted with an ethyl acetate/isopropanol/water (60/30/10 vol/vol) solvent system.…”

“…37,38 ESI/MS/MS analyses of endogenous GlcCer and GalCer molecular species (C18-Sphingoid Base) were performed on a Thermo Fisher TSQ Quantum Triple Quadrupole Mass Spectrometer operating in an MRMpositive ionization mode using a modified version. 38 Briefly, cell pellets corresponding to about 1-3310 6 cells were fortified with synthetic and not natural internal standards (ISs: C8-GluCer, C8-GalCer, C12-GluCer, and C12-GalCer; Avanti Polar Lipids, Inc., Alabaster, AL) and extracted with an ethyl acetate/isopropanol/water (60/30/10 vol/vol) solvent system. After evaporation and reconstitution in 100 ml methanol, samples were injected on the Supercritical Fluid Chromatography/Mass Spectrometry Berger Minigram/TSQ Quantum SFC/MS System and gradient eluted from a Berger Silica 60 A 6-mm particle, 4.63250-mm column with a 1.0 mM methanolic ammonium formate/2 mM aqueous ammonium formate mobile phase system.…”

Apolipoprotein M modulates erythrocyte efflux and tubular reabsorption of sphingosine-1-phosphate

“…Lipids were extracted with 1 ml of an organic solution consisting of ethyl acetate/2-propanol (6:1, v/v) and 50 l of concentrated formic acid was added for phase separation ( 25 ). The upper organic phase was separated and evaporated to dryness under a stream of nitrogen.…”