Identification of isobaric lyso-phosphatidylcholines in lipid extracts of gilthead sea bream (Sparus aurata) fillets by hydrophilic interaction liquid chromatography coupled to high-resolution Fourier-transform mass spectrometry

Abstract

The numerous and varied biological roles of phosphatidylcholines (PC) and lysophosphatidylcholines (LPC) have fueled a great demand for technologies that enable rapid, in-depth structural examination of these lipids in foodstuffs. Here, we describe the capabilities of a newly configured combination of high-efficiency liquid chromatography and high-resolution/accuracy Fourier-transform mass spectrometry with electrospray ionization (LC-ESI-FTMS), designed for lipidomics applications that require the identification of PC in their lyso forms. The devised strategy, involving a separation by hydrophilic interaction liquid chromatography (HILIC) on spherical, fused-core ultrapure silica particles (2.7 μm) of a narrow-bore column (2.1 mm i.d.), enabled the identification of as many as 71 LPC species in the lipid extracts of gilthead sea bream (Sparus aurata) fillets. In this way, LPC as proton (43) and sodium (28) adducts, i.e., [M + H](+) and [M + Na](+) ions (with M representing the zwitterionic form), were identified. In several cases, the extremely high (sub-ppm) mass accuracy and the high chromatographic efficiency available with the adopted instrumentation enabled the distinction of isobaric and closely eluting LPC species. Informative tandem mass spectra, based on high-energy collision induced dissociation (HCD), were also obtained, thus distinguishing regioisomeric LPC species (i.e., LPC differing only for the location of the residual side chain on the glycerol backbone) and between proton and sodium adducts. Graphical Abstract Extracted Ion Current chromatogram (XIC) obtained for the m/z value 568.339, showing the presence of two regioisomeric Lysophosphatidylcholines. The corresponding high collisional energy tandem MS spectra, obtained using a HCD cell, are shown as insets.

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

“…Although the 2D method is comprehensive in coverage of lipids, it will increase substantially the time for analysis, which is not feasible when hundreds of samples need to be analyzed in a lipidomics study. Recently, one dimension HILIC–ESI–MS method has been used for analysis of phospholipids, good separation of lysophopholipids was achieved, while co-elution of other different lipid classes, like PS and PE, was also observed [31]. Although its performance has greatly improved over other HILIC-based lipid separations, its capability in complete separation of phospholipid classes is less desirable compared with a normal phase column, where no co-elution was achieved using a Chromolith–HPLC–ESI–MS [32].…”

“…Similar results to those obtained previously can be achieved with the present method while only using one dimensional reversed phase analysis. Most recently, one dimension HILIC–ESI–MS coupled with HCD–MS/MS was used for the identification of regioisomeric species of LPCs [31]. However, the fragmentation patterns reported by those authors are different from the ones obtained in the present work; for example, neither the m/z 104.11 for the sn-2 LPC nor the [M+H-(P-Chol)] + ion for the sn-1 LPC were obtained in their study.…”

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

“…HILIC was not chosen for the current study because of, as mentioned before, its inability in class separation of nonpolar lipids [6]. Monolithic silica column has been previously used successfully in the separation of different phospholipid classes [21] and also in the study of nonpolar and polar lipids in marine zooplankton [14].…”

“…Same is true for the analysis of lysophospholipid species using this 2D method. Clinically, it is important to accurately identify and quantify lysophospholipids, since it has been demonstrated recently that they are related to cancer and other pathological conditions [6, 30, 31], In addition, some species, like cholesterol esters, were only identified when the samples were analyzed by off-line two-dimensional method. Moreover, a much higher number of TG has been identified using fractionation, which can be attributed to the reduced ion suppression effect in the fractionated samples; in contract, DG and CL species co-eluted with TG in the RPLC chromatogram of unfractionated sample.…”

“…Therefore, HILIC has been mainly used in the analysis of different classes of phospholipids, demonstrating good performance in some methods found in the literature [5], while presenting problems of co-elution of certain classes, like PS and PE, in others [6]. Significant improvement in class-based lipid separation has been achieved recently when hybrid silica stationary phase column was employed in ultrahigh-performance supercritical fluid chromatography (UHPSFC), where both non polar and polar lipid classes can be well separated [7].…”