Lipids In Complex Samples Research Articles

High-resolution ion mobility based on traveling wave structures for lossless ion manipulation resolves hidden lipid features

High-resolution ion mobility (resolving power > 200) coupled with mass spectrometry (MS) is a powerful analytical tool for resolving isobars and isomers in complex samples. High-resolution ion mobility is capable of discerning additional structurally distinct features, which are not observed with conventional resolving power ion mobility (IM, resolving power ~ 50) techniques such as traveling wave IM and drift tube ion mobility (DTIM). DTIM in particular is considered to be the “gold standard” IM technique since collision cross section (CCS) values are directly obtained through a first-principles relationship, whereas traveling wave IM techniques require an additional calibration strategy to determine accurate CCS values. In this study, we aim to evaluate the separation capabilities of a traveling wave ion mobility structures for lossless ion manipulation platform integrated with mass spectrometry analysis (SLIM IM-MS) for both lipid isomer standards and complex lipid samples. A cross-platform investigation of seven subclass-specific lipid extracts examined by both DTIM-MS and SLIM IM-MS showed additional features were observed for all lipid extracts when examined under high resolving power IM conditions, with the number of CCS-aligned features that resolve into additional peaks from DTIM-MS to SLIM IM-MS analysis varying between 5 and 50%, depending on the specific lipid sub-class investigated. Lipid CCS values are obtained from SLIM IM (TW(SLIM)CCS) through a two-step calibration procedure to align these measurements to within 2% average bias to reference values obtained via DTIM (DTCCS). A total of 225 lipid features from seven lipid extracts are subsequently identified in the high resolving power IM analysis by a combination of accurate mass-to-charge, CCS, retention time, and linear mobility-mass correlations to curate a high-resolution IM lipid structural atlas. These results emphasize the high isomeric complexity present in lipidomic samples and underscore the need for multiple analytical stages of separation operated at high resolution.Graphical abstract

Ultrahigh-performance supercritical fluid chromatography for intraclass separation of lipids: Investigation of general principles

Powerful chromatographic techniques are required for lipidomic analyses due to the extreme complexity of natural lipidomes. In the past few years, ultrahigh-performance supercritical fluid chromatography (UHPSFC) has proven to be a good alternative to conventional LC methods for comprehensive lipidomic analysis. The goal of this work was to study UHPSFC intraclass separation of lipids according to the fatty acyl composition. The effects of column chemistry, mobile phase composition and gradient, flow rate, back pressure, temperature, and column coupling on intraclass separation of lipids were widely investigated and discussed. In general, UHPSFC exhibited interclass selectivity together with intraclass separation of lipids according to their total number of double bonds and acyl chain lengths. Moreover, separations of diacylglycerol and lysophosphatidylcholines regioisomers were achieved in some cases. The nature of the stationary phase showed the most prominent effect on UHPSFC intraclass selectivity, while other chromatographic conditions were used for partial improvement in resolution of lipid species. An octadecyl-based stationary phase showed excellent separation of nonpolar lipid species, including triacylglycerol isobars; however, it provided poor peak shapes and limited retention time reproducibility for polar lipids. Diol- and 1-aminoanthracene-based columns provided the best inter- and intraclass resolution of most lipids. The main benefit for UHPSFC separation of complex lipid samples is the combination of the acyl chain/double bond intraclass separation of lipids with excellent lipid class selectivity, which can facilitate mass spectrometry detection and quantitation of trace species without ion suppression effects.

Regio- and Stereospecific Analysis of Triacylglycerols—A Brief Overview of the Challenges and the Achievements

The efforts to reveal, in detail, the molecular and intramolecular structures of one of the main lipid classes, namely, triacyl-sn-glycerols, which are now known to affect their specific and important role in all living organisms, are briefly overviewed. Some milestones of significance in the gradual but continuous development and improvement of the analytical methodology to identify the triacylglycerol regio- and stereoisomers in complex lipid samples are traced throughout the years: the use of chromatography based on different separation principles; the improvements in the chromatographic technique; the development and use of different detection techniques; the attempts to simplify and automatize the analysis without losing the accuracy of identification. The spectacular recent achievements of two- and multidimensional methods used as tools in lipidomics are presented.

Matrix-assisted ionization Fourier transform mass spectrometry for the analysis of lipids.

Assessing the utility of vacuum matrix-assisted ionization (MAI) for the direct and rapid analysis of lipids in complex samples with emphasis on bacterial taxonomy. Matrix-assisted ionization Fourier transform mass spectrometry (MAI-FTMS) was used to characterize polar and non-polar lipids in mixtures. For non-polar lipid triacylglycerols (TAGs), MAI-FTMS produced lipid-specific ions for eight different edible oils and allowed these oils to be identified based on their MAI-FTMS profiles. For polar lipids from bacteria, MAI-FTMS of crude lipid extracts allowed taxonomic identification of eight blind-coded samples based on taxonomy-specific phospholipid profiles. MAI produced results comparable and complementary to benchmark MALDI and ESI methods currently used for characterization of polar and non-polar lipids in the same mixtures. The newly developed MAI technique is a rapid, simple and complementary method for the characterization of polar and non-polar lipids in complex mixtures.

Optimization of Electrospray Ionization Source Parameters for Lipidomics To Reduce Misannotation of In-Source Fragments as Precursor Ions.

Lipidomics requires the accurate annotation of lipids in complex samples to enable determination of their biological relevance. We demonstrate that unintentional in-source fragmentation (ISF, common in lipidomics) generates ions that have identical masses to other lipids. Lysophosphatidylcholines (LPC), for example, generate in-source fragments with the same mass as free fatty acids and lysophosphatidylethanolamines (LPE). The misannotation of in-source fragments as true lipids is particularly insidious in complex matrixes since most masses are initially unannotated and comprehensive lipid standards are unavailable. Indeed, we show such LPE/LPC misannotations are incorporated in the data submitted to the National Institute of Standards and Technology (NIST) interlaboratory comparison exercise. Computer simulations exhaustively identified potential misannotations. The selection of in-source fragments of highly abundant lipids as features, instead of the correct recognition of trace lipids, can potentially lead to (i) missing the biologically relevant lipids (i.e., a false negative) and/or (ii) incorrect assignation of a phenotype to an incorrect lipid (i.e., false positive). When ISF is not eliminated in the negative ion mode, ∼40% of the 100 most abundant masses corresponding to unique phospholipids measured in plasma were artifacts from ISF. We show that chromatographic separation and ion intensity considerations assist in distinguishing precursor ions from in-source fragments, suggesting ISF may be especially problematic when complex samples are analyzed via shotgun lipidomics. We also conduct a systematic evaluation of electrospray ionization (ESI) source parameters on an Exactive equipped with a heated electrospray ionization (HESI-II) source with the objective of obtaining uniformly appropriate source conditions for a wide range of lipids, while, at the same time, reducing in-source fragmentation.

Identification of oxidized phospholipids in bronchoalveolar lavage exposed to low ozone levels using multivariate analysis

Chemical reactions with unsaturated phospholipids in the respiratory tract lining fluid have been identified as one of the first important steps in the mechanisms mediating environmental ozone toxicity. As a consequence of these reactions, complex mixtures of oxidized lipids are generated in the presence of mixtures of non-oxidized naturally occurring phospholipid molecular species, which challenge methods of analysis. Untargeted mass spectrometry and statistical methods were employed to approach these complex spectra. Human bronchoalveolar lavage (BAL) was exposed to low levels of ozone, and samples with and without derivatization of aldehydes were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry. Data processing was carried out using principal component analysis (PCA). Resulting PCA scores plots indicated an ozone dose-dependent increase, with apparent separation between BAL samples exposed to 60ppb ozone and non-exposed BAL samples as well as a clear separation between ozonized samples before and after derivatization. Corresponding loadings plots revealed that more than 30 phosphatidylcholine (PC) species decreased due to ozonation. A total of 13 PC and 6 phosphatidylglycerol oxidation products were identified, with the majority being structurally characterized as chain-shortened aldehyde products. This method exemplifies an approach for comprehensive detection of low-abundance, yet important, components in complex lipid samples.

Raman spectroscopy of lipids: a review

Raman spectra of 35 lipids are presented and analyzed in this work. Selected compounds, i.e. saturated/unsaturated fatty acids, triacylglycerols, cholesterol, cholesteryl esters and phospholipids, were chosen to review key lipids involved in cardiovascular disease development. Differences in Raman signatures both between diverse groups of lipids as well as various members of the same family are investigated in detail in order to elucidate marker features enabling detection and discrimination of lipids in complex samples, particularly of biological origin. This work complements our previous review on important biomolecules, i.e. proteins, and presents a comprehensive database of Raman spectra of naturally occurring lipids. Copyright © 2014 John Wiley & Sons, Ltd.

Aliphatic chain length by isotropic mixing (ALCHIM): determining composition of complex lipid samples by ¹H NMR spectroscopy.

Quantifying the amounts and types of lipids present in mixtures is important in fields as diverse as medicine, food science, and biochemistry. Nuclear magnetic resonance (NMR) spectroscopy can quantify the total amounts of saturated and unsaturated fatty acids in mixtures, but identifying the length of saturated fatty acid or the position of unsaturation by NMR is a daunting challenge. We have developed an NMR technique, aliphatic chain length by isotropic mixing, to address this problem. Using a selective total correlation spectroscopy technique to excite and transfer magnetization from a resolved resonance, we demonstrate that the time dependence of this transfer to another resolved site depends linearly on the number of aliphatic carbons separating the two sites. This technique is applied to complex natural mixtures allowing the identification and quantification of the constituent fatty acids. The method has been applied to whole adipocytes demonstrating that it will be of great use in studies of whole tissues.

Locating double bonds in lipids – New approaches to the use of ozonolysis

AbstractSince the location of double bonds within a lipid molecule is critical to its biological function, methods that reveal details of this structural arrangement are of importance. Ozone can react with double bonds resulting in ozonolysis products that have masses characteristic of the exact location of these double bonds within fatty acid residues. This paper describes the use of the ozonolysis reaction for the assignment of double bond positions, with a focus on the recent developments in coupling ozonolysis reactions in‐situ and in‐line with mass spectrometry, even for use with complex lipid samples.

Non-aqueous microchip electrophoresis for characterization of lipid biomarkers

In vivo measurements of lipid biomarkers are hampered by their low solubility in aqueous solution, which limits the choices for molecular separations. Here, we introduce non-aqueous microchip electrophoretic separations of lipid mixtures performed in three-dimensional hybrid nanofluidic/microfluidic polymeric devices. Electrokinetic injection is used to reproducibly introduce discrete femtolitre to picolitre volumes of charged lipids into a separation microchannel containing low (100 μM-10 mM) concentration tetraalkylammonium tetraphenylborate background electrolyte (BGE) in N-methylformamide, supporting rapid electro-osmotic fluid flow in polydimethylsiloxane microchannels. The quality of the resulting electrophoretic separations depends on the voltage and timing of the injection pulse, the BGE concentration and the electric field strength. Injected volumes increase with longer injection pulse widths and higher injection pulse amplitudes. Separation efficiency, as measured by total plate number, N, increases with increasing electric field and with decreasing BGE concentration. Electrophoretic separations of binary and ternary lipid mixtures were achieved with high resolution (R s ∼ 5) and quality (N > 7.7 × 10(6) plates m(-1)). Rapid in vivo monitoring of lipid biomarkers requires high-quality separation and detection of lipids downstream of microdialysis sample collection, and the multilayered non-aqueous microfluidic devices studied here offer one possible avenue to swiftly process complex lipid samples. The resulting capability may make it possible to correlate oxidative stress with in vivo lipid biomarker levels.

On the Combined Application of Iatroscan TLC-FID and GC-FID to Identify Total, Neutral, and Polar Lipids and Their Fatty Acids Extracted from Foods

An efficient separation and quantification of the individual neutral and polar lipid classes and their constituent fatty acids was achieved by the combination of two different detection techniques: Iatroscan TLC-FID and GC-FID. The solvent composition and ratio of development system, the sample size, the fidelity, and precision were tested in order to estimate the effectiveness of separation of individual neutral and polar lipid classes and the quantitative reproducibility of the Iatroscan TLC-FID technique. GC-FID method, with a high-quality capillary column, allowed sensitive and reproducible fatty acid qualitative and quantitative analyses, separation of fatty acid structural isomers (e.g., n-C16:0, iso-C16:0 and anteiso-C16:0), positional isomers (e.g., C18:1ω-9 and C18:1ω-7), geometrical isomers (cis-trans), and homologues (e.g., C16:0, C17:0, C18:0, etc.) in standards and complex lipid samples. Seventeen (17) lipid classes and fifty-two (52) saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids were identified and quantified, respectively, in samples of standard lipid and fatty acid mixtures, simulating the composition of natural lipids and their fatty acid methyl esters in common foods. The wide number of applications establishes this combination of Iatroscan TLC-FID and GC-FID methods as a powerful tool for lipid class and fatty acid analysis of any fat origin.

Characterization of lipids in complex samples using comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry

Most lipids are a complex mixture of classes of compounds such as fatty acids, fatty alcohols, diols, sterols and hydroxy acids. In this study, the suitability of comprehensive two-dimensional gas chromatography coupled to a time-of-light mass spectrometer is studied for lipid characterization in complex samples. With lanolin, a refined wool wax, as test sample, it is demonstrated that combined methylation plus silylation is the preferred derivatization procedure to achieve (i) high-quality GC × GC separation and (ii) easily recognizable ordered structures in lipid analysis. Optimization of the GC × GC column combination, the influence of the temperature programme on the quality of the separation, and the potential and limitations of automated TOF-MS-based identification are discussed. The combined power of a 2D separation, ordered structures and MS detection is illustrated by the identification of several minor sample constituents.

Near Infrared (NIR) Spectroscopy in the Undergraduate Chemistry Curriculum

The power of NIR spectroscopy is best and most often realized in the study of compounds containing O-H, N-H and C-H bonds. For example, the content of protein, moisture, carbohydrates, and lipids in complex samples can be determined with the use of NIR spectroscopy, without the need for time-consuming extraction steps.

Silver ion high-performance liquid chromatography and gas chromatography—mass spectrometry in the analysis of complex fatty acid mixtures: application to marine invertebrates

High-performance liquid chromatography on a stable silver ion column permitted the isolation of clean fractions differing in degree of unsaturation, with zero to six double bonds, of the methyl ester derivatives of fatty acids from complex lipid samples. By a combination of gas chromatographic analysis of the methyl esters on capillary columns coated with polar phases, and gas chromatography-mass spectrometry of the picolinyl ester derivatives, separated on non-polar phases, most components can be identified. In order to demonstrate the methodology, fatty acids from the Black Sea invertebrates Mytilus galloprovincialis and Rapana thomasiana were separated into 41 and 46 defined components, respectively (58 different fatty acids in total), and full quantitative analyses were achieved of most constituents present at levels of about 0.1% of the total and above. The fatty acids found included mono- and multi-methyl branched isomers, mono- and polyunsaturated fatty acids of the ( n-1), ( n-3), ( n-4), ( n-5), ( n-6), ( n-7), ( n-8), ( n-9), ( n-11) and ( n-13) families, and dienoic acids with several methylene groups between the double bonds, ranging in chain length from C 13 to C 22.

Gradient-thickness thin-layer chromatography for the isolation and analysis of trace amounts of free fatty acids in large lipid samples

A thin-layer chromatographic method for quantitative isolation of free fatty acids is described. This method appears to be more satisfactory than existing methods in offering the combination of advantages of specificity, simplicity, rapidity, reproducibility, accuracy, high sensitivity, and applicability as a preparative technique. The method involves chromatography on a thin-layer plate on which the layer of Silica Gel G decreases linearly in thickness from 1000 micro at the base to 125 micro at the upper end. This gradient-thickness design allows the separation and densitometric quantitation of very small traces of free fatty acids from relatively large and complex lipid samples in a single chromatographic step. The method has been shown to be applicable directly to the crude total lipid extracts of several mammalian tissues. It appears to generate little if any artifactual free fatty acids from the breakdown of complex lipids, in contrast to the undesirable behavior of silicic acid columns in this respect. Gradient-thickness thin-layer chromatography promises to be useful for the quantitative isolation of trace amounts not only of other types of lipids but also of classes of compounds other than lipids.