As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

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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.

S phingolipid (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 sphingosine1-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.

Novel isosteric analogs of the ceramidase inhibitors (1S,2R)-N-myristoylamino-phenylpropanol-1 (d-e-MAPP) and (1R,2R)-N-myristoylamino-4'-nitro-phenylpropandiol-1,3 (B13) with modified targeting and physicochemical properties were developed and evaluated for their effects on endogenous bioactive sphingolipids: ceramide, sphingosine, and sphingosine 1-phosphate (Cer, Sph, and S1P) in MCF7 cells as determined by high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Time- and dose-response studies on the effects of these compounds on Cer species and Sph levels, combined with structure-activity relationship (SAR) data, revealed 4 distinct classes of analogs which were predominantly defined by modifications of the N-acyl-hydrophobic interfaces: N-acyl-analogs (class A), urea-analogs (class B), N-alkyl-analogs (class C), and omega-cationic-N-acyl analogs (class D). Signature patterns recognized for two of the classes correspond to the cellular compartment of action of the new analogs, with class D acting as mitochondriotropic agents and class C compounds acting as lysosomotropic agents. The neutral agents, classes A and B, do not have this compartmental preference. Moreover, we observed a close correlation between the selective increase of C(16)-, C(14)-, and C(18)-Cers and inhibitory effects on MCF7 cell growth. The results are discussed in the context of compartmentally targeted regulators of Sph, Cer species, and S1P in cancer cell death, emphasizing the role of C(16)-Cer. These novel analogs should be useful in cell-based studies as specific regulators of Cer-Sph-S1P inter-metabolism, in vitro enzymatic studies, and for therapeutic development.