Facile determination of sphingolipids under alkali condition using metal-free column by LC-MS/MS.

Extraction and analysis of sphingolipids from biological samples is a critical step in lipidomics, especially for minor species such as sphingoid bases and sphingosine-1-phosphate. Although several liquid chromatography-mass spectrometry methods enabling the determination of sphingolipid molecular species have been reported, they were limited in analytical sensitivity and reproducibility by causing significant peak tailing, especially by the presence of phosphate groups, and most of the extraction techniques are laborious and do not cover a broad range of sphingolipid metabolites. In this study, we developed a rapid single-phase extraction and highly sensitive analytical method for the detection and quantification of sphingolipids (including phosphates) comprehensively using liquid chromatography-triple quadruple mass spectrometry. After validating the reliability of the method, we analyzed the intestinal tissue sphingolipids of germ-free (GF) and specific pathogen-free (SPF) mice and found significantly higher levels of free sphingoid bases and sphingosine-1-phosphate in the GF condition as compared to the SPF condition. This method enables a rapid extraction and highly sensitive determination of sphingolipids comprehensively at low femtomolar ranges. Graphical abstract Diagrammatic comparision of sphingolipid (phosphates) analysis between conventional and this method.

Sphingolipid analysis by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

Sphingolipid (SPL) metabolism (Fig. 1) serves a key role in the complex mechanisms regulating cellular stress responses to environment. Several SPL metabolites, especially ceramide (Cer), sphingosine (Sph) and sphingosinel-phosphate (S1P) act as key bioactive molecules governing cell growth and programmed cell death (Fig. 2). Perturbations in sphingolipids of one type may enhance or interfere with the action of another. To monitor changes in SPL composition therefore, reliable analytical methods are necessary. Here we present the liquid chromatography tandem mass spectrometry (LC-MS/MS) approach for simultaneous qualitative and quantitative monitoring of SPL components (classes and molecular species) in biological material as an effective tool to study sphingolipid signaling events. The LC-MS/MS methodology is the only available technique that provides high specificity and sensitivity, along with a wealth of structural identification information.

Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers.

Sphingolipids are a highly diverse category of bioactive compounds. This article describes methods that have been validated for the extraction, liquid chromatographic (LC) separation, identification and quantitation of sphingolipids by electrospray ionization, tandem mass spectrometry (ESI-MS/MS) using triple quadrupole (QQQ, API 3000) and quadrupole-linear-ion trap (API 4000 QTrap, operating in QQQ mode) mass spectrometers. Advantages of the QTrap included: greater sensitivity, similar ionization efficiencies for sphingolipids with ceramide versus dihydroceramide backbones, and the ability to identify the ceramide backbone of sphingomyelins using a pseudo-MS3 protocol. Compounds that can be readily quantified using an internal standard cocktail developed by the LIPID MAPS Consortium are: sphingoid bases and sphingoid base 1-phosphates, more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides, and these complex sphingolipids with dihydroceramide backbones. With minor modifications, glucosylceramides and galactosylceramides can be distinguished, and more complex species such as sulfatides can also be quantified, when the internal standards are available. LC ESI-MS/MS can be utilized to quantify a large number of structural and signaling sphingolipids using commercially available internal standards. The application of these methods is illustrated with RAW264.7 cells, a mouse macrophage cell line. These methods should be useful for a wide range of focused (sphingo)lipidomic investigations.