Enhanced lipid isomer separation in human plasma using reversed-phase UPLC with ion-mobility/high-resolution MS detection

Abstract

This article is available online at http://www.jlr.orgLipids are the building blocks of all cell membranes and are thus essential for various biological functions varying from membrane traffi cking to signal transduction. Disorders in lipid metabolism play a key role in various diseases including cardiovascular disease, cancer, diabetes mellitus, and infl ammation ( 1, 2 ). Lipids have been classifi ed into eight categories (fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides) by the International Committee for the Classifi cation and Nomenclature of Lipids in conjunction with the LIPID MAPS consortium, based on their chemically distinct functional backbones and hydrophobic nature ( 3, 4 ).As the study of lipids attracts more and more attention, lipidomics, as a branch of metabolomics, is a quickly emerging fi eld. It has been defi ned as "the full characterization of lipid molecular species and of their biological roles with respect to expression of proteins involved in lipid metabolism and function, including gene regulation" ( 5 ).In order to achieve the goal of full characterization, different MS-based approaches have been described. Some groups describe shotgun lipidomics using direct infusion into the mass spectrometer ( 6-11 ). Direct infusion MS has the advantage of short analysis times and consuming very small amounts of sample. However, structural elucidation of lipid isomers is not possible. In order to elucidate structures Abstract An ultraperformance LC (UPLC) method for the separation of different lipid molecular species and lipid isomers using a stationary phase incorporating charged surface hybrid (CSH) technology is described. The resulting enhanced separation possibilities of the method are demonstrated using standards and human plasma extracts. Lipids were extracted from human plasma samples with the Bligh and Dyer method. Separation of lipids was achieved on a 100 × 2.1 mm inner diameter CSH C 18 column using gradient elution with aqueous-acetonitrile-isopropanol mobile phases containing 10 mM ammonium formate/0.1% formic acid buffers at a fl ow rate of 0.4 ml/min. A UPLC run time of 20 min was routinely used, and a shorter method with a 10 min run time is also described. The method shows extremely stable retention times when human plasma extracts and a variety of biofl uids or tissues are analyzed [intra-assay relative standard deviation (RSD) <0.385% and <0.451% for 20 and 10 min gradients, respectively (n = 5); interassay RSD <0.673% and <0.763% for 20 and 10 min gradients, respectively (n = 30)]. The UPLC system was coupled to a hybrid quadrupole orthogonal acceleration time-of-fl ight mass spectrometer, equipped with a traveling wave ion-mobility cell. Besides demonstrating the separation for different lipids using the chromatographic method, we demonstrate the use of the ion-mobility MS platform for the structural elucidation of lipids. The method can now be used to elucidate structures of a wide variety of lipid...

Combining liquid chromatography with ozone-induced dissociation for the separation and identification of phosphatidylcholine double bond isomers

“…n--9 elutes before n--12, Figure 2a). This observation is consistent with previous reports of separation of double bond positional isomers and evidence from new column technologies suggests further improvements in isomer separation can be achieved [17,19,18]. Full structure elucidation of complex lipids however, also requires separation and/or mass spectral differentiation of isomers that differ in (i) the position of acyl chains on the glycerol backbone (i.e., sn--positional isomers) and the stereochemistry about the double bond(s) (i.e., cis and trans).…”

“…Prior observation of the behaviour of unsaturated phosphatidylcholines on reversed phase columns suggests that the peaks eluting at 49.0 and 50.4 min correspond to the isomers PC 18:1(n--9)/18:1(n--9) and PC 18:1(n--12)/18:1(n--12), respectively [17,19,18]. Integration of OzID scans obtained across each of the chromatographic peaks gave the OzID mass spectra shown in bonds closer to the methyl terminus of lipid acyl chains has previously been noted [24].…”

“…In contrast, analytical separations of phospholipid double bond positional isomers -where the acyl chains are themselves isomeric -by reversed--phase LC have proven more challenging. Prepared mixtures of two synthetic phosphatidylcholine double bond positional isomers, PC 18:1(n--9)/18:1(n--9) and PC 18:1(n--12)/18:1(n--12), have been successfully separated by reversed--phase LC using a C 8 column and, more recently, using charged surface hybrid C 18 columns [17,18]. While these analytical demonstrations are important, very few studies have differentiated between endogenous double bond positional isomers in complex biological extracts.…”

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

“…On the other hand, Damen et al [28] demonstrated the superior separation of different isomers using the CSH (charged surface hybrid) C18 column in comparison with the HSS T3. Despite its bigger particle size (2.6 μm), Accucore C30 column showed higher performance than the other three columns with sub 2 μm particles.…”

Ion Mobility in Clinical Analysis: Current Progress and Future Perspectives

“…Furthermore, unsaturated phosphatidylcholines have been separated with TWIMS-MS on the basis of differences in chain length and double bond number/position (67 ). More recently, LC-TWIMS-MS allowed resolution of coeluting lipid isomers in human plasma extracts, showing the potential for this technique to improve clinical LC-MS methods both qualitatively and quantitatively (68 ). Collection of ion mobility-derived CCS values can further improve lipidomics specificity, especially with complex biological samples or when performing MALDI imaging studies that offer no chromatographic separation (69 ).…”