Comprehensive untargeted lipidomic analysis using core–shell C30 particle column and high field orbitrap mass spectrometer

Abstract

The goal of untargeted lipidomics is to have high throughput, yet comprehensive and unambiguous identification and quantification of lipids. Novel stationary phases in LC separation and new mass spectrometric instruments capable of high mass resolving power and faster scanning rate are essential to achieving this goal. In this work, 4 reversed phase LC columns coupled with a high field quadrupole orbitrap mass spectrometer (Q Exactive HF) were thoroughly compared using complex lipid standard mixture and rat plasma and liver samples. A good separation of all lipids was achieved in 24 min of gradient. The columns compared include C30 and C18 functionalization on either core–shell or totally porous silica particles, with size ranging from 1.7 to 2.6 μm. Accucore C30 column showed the narrowest peaks and highest theoretical plate number, and excellent peak capacity and retention time reproducibility (<1% standard deviation). As a result, it resulted in 430 lipid species identified from rat plasma and rat liver samples with highest confidence. The high resolution offered by the up-front RPLC allowed discrimination of cis/trans isomeric lipid species, and the high field orbitrap mass spectrometer afforded the clear distinction of isobaric lipid species in full scan MS and the unambiguous assignment of sn-positional isomers for lysophospholipids in MS/MS. Taken together, the high efficiency LC separation and high mass resolving MS analysis are very promising tools for untargeted lipidomics analysis.

Off-line mixed-mode liquid chromatography coupled with reversed phase high performance liquid chromatography-high resolution mass spectrometry to improve coverage in lipidomics analysis

“…In both samples, a higher number of 1,2-DG species were identified in comparison with 1,3-DG. No MG species were found in either sample and no CL species were found in rat plasma, which is in accordance with our previous work [3]. So species were co-eluted with Cer in mixed-mode LC, this is likely caused by the similarities of their molecule head group.…”

“…The same strategy was applied to the fractions I, J and K because there are no isobaric species present in the same fraction. In the case of the fraction M where regioisomers of LysoPC were collected, this also did not pose a problem since the RPLC-MS/MS method permits the identification of positional isomers for this lipid class, as was reported previously [3]. …”

“…The RPLC method used has demonstrated good performance in lipidomic analysis of total lipids [3]. The fractions collected using the mixed-mode LC method were completely dried before reconstitution and injection onto the RPLC column.…”

“…The different lipid fractions were analyzed according to our previous work [3]. A Vanquish UHPLC system (ThermoFisher Scientific) with a binary pump and an autosampler was used for this study, which was online coupled to a Q Exactive HF (QEHF) hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific).…”

“…Core-shell particles have become more popular in recent years for their reduced resistance to mass transfer and high particle uniformity, overcoming the ultrahigh backpressure resulted from the porous sub-2 μm particles [2]. Recently, we applied a C30 column to the analysis of complex lipid samples and compared it with several C18 columns, the results showed that it has excellent peak capacity and retention time reproducibility, besides of allowing the highest number of lipids identified in rat plasma and liver samples [3]. …”