New methods are needed for global lipid profiling due to the complex chemical structures and diverse physicochemical properties of lipids. Herein we introduce a robust data workflow to unambiguously select lipid features from serum ether extracts by multisegment injection−nonaqueous capillary electrophoresis−mass spectrometry (MSI−NACE−MS). An iterative three-stage screening strategy is developed for nontargeted lipid analyses when using multiplexed electrophoretic separations coupled to an Orbitrap mass analyzer under negative ion mode. This approach enables the credentialing of 270 serum lipid features annotated based on their accurate mass and relative migration time, including 128 ionic lipids reliably measured (median CV ≈ 13%) in most serum samples (>75%) from nonalcoholic steatohepatitis (NASH) patients (n = 85). A mobility map is introduced to classify charged lipid classes over a wide polarity range with selectivity complementary to chromatographic separations, including lysophosphatidic acids, phosphatidylcholines, phosphatidylinositols, phosphatidylethanolamines, and nonesterified fatty acids (NEFAs). Serum lipidome profiles were also used to differentiate high-from low-risk NASH patients using a k-means clustering algorithm, where elevated circulating NEFAs (e.g., palmitic acid) were associated with increased glucose intolerance, more severe liver fibrosis, and greater disease burden. MSI−NACE−MS greatly expands the metabolome coverage of conventional aqueous-based CE−MS protocols and is a promising platform for large-scale lipidomic studies.

Orthogonal separation techniques coupled to high-resolution mass spectrometry (MS) are required for characterization of the human lipidome given its inherent chemical and structural complexity. However, electrophoretic separations remain largely unrecognized in contemporary lipidomics research as compared to various chromatographic and ion mobility methods. Herein, we introduce a novel derivatization protocol based on 3-methyl-1-p-tolyltriazene (MTT) as a safer alternative to diazomethane for quantitative phospholipid methylation (~ 90%), which enables their rapid analysis by multisegment injection-nonaqueous capillary electrophoresis-mass spectrometry (MSI-NACE-MS). Isobaric interferences and ion suppression effects were minimized by performing an initial reaction using 9-fluorenylmethyoxycarbonyl chloride with a subsequent back extraction in hexane. This charge-switch derivatization strategy expands lipidome coverage when using MSI-NACE-MS under positive ion mode with improved resolution, greater sensitivity and higher throughput (~ 3.5 min/sample), notably for zwitter-ionic phospholipids that are analyzed as their cationic phosphate methyl esters. Our method was validated by analyzing methyl-tert-butyl ether extracts of NIST SRM-1950 human plasma, which allowed for a direct comparison of 53 phosphatidylcholine and 30 sphingomyelin species previously reported in an inter-laboratory lipidomics harmonization study. The potential for reliable plasma phospholipid quantification by MSI-NACE-MS via a serial dilution of NIST-SRM-1950 was also demonstrated based on estimation of relative response factors using their reported consensus concentrations. Also, lipid identification was supported by modeling characteristic changes in the electrophoretic mobility for cationic phospholipids in conjunction with MS/MS. Overall, this work offers a practical derivatization protocol to expand lipidome coverage in CE-MS beyond the analysis of hydrophilic/polar metabolites under aqueous buffer conditions, which may also prove useful in shotgun and LC-MS lipidomic applications.