Improvement of a solid phase extraction method for analysis of lipid fractions in muscle foods

The most used method for muscle lipid fractionation into major lipid classes was modified for improving its separation efficiency. Extracted lipids from a masseter muscle of one Iberian pig were separated into neutral lipids (NL), free fatty acids (FFA) and polar lipids (PL) using aminopropyl minicolumns, following the extensively used method of Kaluzny et al. [1] (old method-OM-) and a method based on that, developed by Pinkart et al. [2] with some (modifications modified method–MM). Obtained lipid classes were further analysed by TLC and lipid fractions were identified. TLC evidenced the presence of a certain amount of PL in the NL fraction obtained with the OM. On the other hand, using the MM only an almost undetectable presence of PL was evidenced in the NL fraction. Fatty acid composition of NL, PL and FFA obtained with each method was studied by gas chromatography. Fatty acid profile of NL was strongly influenced by the separation method used. Thus, NL obtained using the OM showed higher amounts of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) and lower of monounsaturated fatty acids (MUFA) than those obtained using the MM. Moreover, NL obtained using the OM showed the presence of fatty alcohols, constituents of phospholipids (PhL) absent or present only in trace amounts in acylglycerols. This profile reflects the coelution of PL in the NL fraction. Fatty acid profile of FFA and PL fractions was also influenced by the solid phase extraction (SPE) method used, but to a lesser extent.     

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 x GC separation and (ii) easily recognizable ordered structures in lipid analysis. Optimization of the GC x 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.     

Accurate and reliable quantification of total microalgal fuel potential as fatty acid methyl esters by <Emphasis Type="Italic">in situ</Emphasis> transesterification

In the context of algal biofuels, lipids, or better aliphatic chains of the fatty acids, are perhaps the most important constituents of algal biomass. Accurate quantification of lipids and their respective fuel yield is crucial for comparison of algal strains and growth conditions and for process monitoring. As an alternative to traditional solvent-based lipid extraction procedures, we have developed a robust whole-biomass in situ transesterification procedure for quantification of algal lipids (as fatty acid methyl esters, FAMEs) that (a) can be carried out on a small scale (using 4–7 mg of biomass), (b) is applicable to a range of different species, (c) consists of a single-step reaction, (d) is robust over a range of different temperature and time combinations, and (e) tolerant to at least 50% water in the biomass. Unlike gravimetric lipid quantification, which can over- or underestimate the lipid content, whole biomass transesterification reflects the true potential fuel yield of algal biomass. We report here on the comparison of the yield of FAMEs by using different catalysts and catalyst combinations, with the acid catalyst HCl providing a consistently high level of conversion of fatty acids with a precision of 1.9% relative standard deviation. We investigate the influence of reaction time, temperature, and biomass water content on the measured FAME content and profile for 4 different samples of algae (replete and deplete Chlorella vulgaris, replete Phaeodactylum tricornutum, and replete Nannochloropsis sp.). We conclude by demonstrating a full mass balance closure of all fatty acids around a traditional lipid extraction process.     

Lipid classes of in vitro cultivar of Pakistani citrus

The in vitro cultivar of citrus contained 18.0% lipids after 12 weeks of germination of seed. The lipid was analyzed by GC procedure for its fatty acid composition. The oil contained seven major fatty acid constituents which were later identified by GC. The oil was further analyzed for its classes by means of thin layer chromatography and gas chromatography. The major portion of the lipid classes comprised of neutral lipids (93.9%) and polar lipids (6.1%). The identified neutral lipids comprised of hydrocarbon (1.4%), wax esters (1.5%), sterol esters (5.2%), triglycerides (52.3%), free fatty acids (1.3%), 1,3-diglycerides (6.0%), 1,2-diglycerides (5.0%), glycol (15.2%), sterols (6.0%), 2-monoglycerides (6.4%), 1-monoglycerides (5.3%) and the identified polar lipids comprised of phosphatidyl ethanolamine (1.8%) phosphatidyl choline (0.9%) lysophosphatidyl ethanolamine (1.8%) and phosphatidyl inositol (1.1%).     

超高效液相色谱-飞行时间-串联质谱法用于大白菜叶片中的脂质成分分析

建立了超高效液相色谱-飞行时间-串联质谱(UPLC-TOF-MS/MS)分析大白菜叶片中脂质的种类、结构、脂肪酸组成并检测其相对含量的方法。以Acquity UPLCTMBEH C8色谱柱为固定相,采用飞行时间质谱全扫描-信息关联采集-子离子扫描(TOF-MS scan-IDA-product ion scan)复合模式实现一次进样分析,同时获得脂质的一级和二级质谱信息。在大白菜叶片中共鉴定得到232种脂质,包含104种磷脂、63种糖脂和65种甘油酯。其中,磷脂、糖脂和甘油酯中的主要成分分别为磷脂酰胆碱、单半乳糖二酰甘油和甘油二酯、甘油三酯。结果表明,该方法具有灵敏度高、准确度高和高通量等优点,为植物脂质的代谢研究提供了可靠的分析技术平台,并为进一步的脂质生物学功能研究奠定了基础。     

Anti-Inflammatory and Anti-Platelet Properties of Lipid Bioactives from Apple Cider By-Products

The valorization of food industry by-products as sources of bioactive compounds is at the forefront of research in functional foods and nutraceuticals. This study focuses on bioactives of apple cider by-products (ACBPs) with putative cardio-protective properties. Total lipids (TLs) were extracted from ACBPs of apple varieties that are low (ACBP1), medium (ACBP2), and high (ACBP3) in tannins and were further separated into polar lipids (PLs) and neutral lipids (NLs). The functionality of these lipid extracts and of their HPLC-derived lipid fractions/PL subclasses were assessed in vitro against human platelet aggregation induced by the thrombotic and inflammatory platelet agonists platelet-activating factor (PAF) and adenosine diphosphate (ADP). The fatty acid profile of PLs and their most bioactive lipid fractions were evaluated by GC–MS analysis. The PL extracts exhibited higher specificity against the PAF-induced platelet aggregation compared to their anti-ADP effects, while TL and NL showed lower bioactivities in all ACBPs. HPLC analysis unveiled that the most bioactive PL from all ACBPs were those in PL fraction 3 containing phosphatidylcholines (PCs). PLs from all ACBPs and their PC bioactives were rich in polyunsaturated fatty acids (PUFAs) and especially in the essential omega-6 (n-6) linoleic acid (LA) and omega-3 (n-3) alpha linolenic acid (ALA), with favorably low values of the n-6/n-3 PUFA ratio, thus providing a rationale for their higher anti-inflammatory bioactivities. Within this study, highly bioactive PL compounds with strong anti-inflammatory and anti-platelet properties were identified in ACBPs, which can be potentially utilized for producing cardio-protective functional foods and/or nutraceuticals.     

Profiling and quantification of Drosophila melanogaster lipids using liquid chromatography/mass spectrometry

We present here the findings of global profiling of Drosophila lipids using liquid chromatography/tandem mass spectrometry (LC/MS/MS) on an LTQ-Orbitrap instrument. In addition, we present a multiple reaction monitoring (LC-MRM) method for the absolute quantification of the major phosphatidylethanolamine (PE) and phosphatidylcholine (PC) lipids of Drosophila. Using both normal- and reversed-phase LC followed by accurate mass analysis and MS/MS on an LTQ-Orbitrap instrument, we evaluated the lipid composition of the fruit fly Drosophila melanogaster. A total of 74 lipid species were identified consisting of glycerphospholipids belonging to the PE, PC, phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PS) classes including several plasmanyl PE species, as well as triacylglycerides, cardiolipins, ceramides, and PE ceramides. Individual PE and PC phospholipids were then quantified using an LC-MRM approach. Reversed-phase chromatography followed by monitoring on a QTrap 4000 instrument of 21 MRM transitions combined with calibration curves constructed using internal standards enabled the absolute quantification of 28 PE and PC lipid species with limits of quantification of 3 and 5 pg/μL, respectively. Internal standards accounted for the differences in ionization efficiencies of PE and PC phospholipids, facilitating more accurate lipid abundance measurements. The method presented here builds on previous Drosophila work by making the quantification of absolute lipid abundance possible and will be of interest to scientists who study variation and changes in the degree of unsaturation, fatty acid carbon length, and head-group concentration among individuals of different genotypes in response to environmental, genetic, or physiological perturbation in small insects. It will also be particularly useful to biologists interested in adaptation and acclimation of cellular membranes in response to thermal heterogeneity.     

Lipid Biomarkers of Lens Aging

Lipids are important structural components of cell membranes and have profound effect on membrane fluidity. Lipid profiling and lipidomics have captured increased attention due to the well-recognized roles of lipids in numerous human diseases. Investigating lipid profiles not only provides insights into the specific roles of lipid molecular species in health and diseases, but can also help in identifying potential preventive or therapeutic biomarkers. Cataract, the loss of transparency of eye lens, is a disease of protein aggregation. There are several factors contributing to the stability in protein conformation. Age-related changes in lipid composition could be a contributing factor for altered protein–lipid interaction leading to protein aggregation and cataract. Keeping this in view, in the present study, fatty acid profiling from different age groups of lenses was carried out, using a freshwater catfish as the model. Total lipids were extracted from lenses of three different age groups of fishes (young, adult, and aged) and fatty acid methyl esters (FAME) were prepared and FAME analysis was carried out using gas chromatography–mass spectrometry. The results showed that three fatty acids viz. heneicosylic acid (C21), docosahexaenoic acid (C22:6), nervonic acid (C24:1) which were not present in the adult lens, appeared in the aged lens. On the other hand, eicosenoic acid (C20:1) present in the adult lens was found to be absent in the aged lens. The appearance or disappearance of these fatty acids can possibly serve as biomarkers of aging lens which is the most vulnerable stage for cataract development.     

Analysis of lipids with desorption atmospheric pressure photoionization‐mass spectrometry (DAPPI‐MS) and desorption electrospray ionization‐mass spectrometry (DESI‐MS)

In this article, the effect of spray solvent on the analysis of selected lipids including fatty acids, fat‐soluble vitamins, triacylglycerols, steroids, phospholipids, and sphingolipids has been studied by two different ambient mass spectrometry (MS) methods, desorption electrospray ionization‐MS (DESI‐MS) and desorption atmospheric pressure photoionization‐MS (DAPPI‐MS). The ionization of the lipids with DESI and DAPPI was strongly dependent on the spray solvent. In most cases, the lipids were detected as protonated or deprotonated molecules; however, other ions were also formed, such as adduct ions (in DESI), [M‐H]⁺ ions (in DESI and DAPPI), radical ions (in DAPPI), and abundant oxidation products (in DESI and DAPPI). DAPPI provided efficient desorption and ionization for neutral and less polar as well as for ionic lipids but caused extensive fragmentation for larger and more labile compounds because of a thermal desorption process. DESI was more suitable for the analysis of the large and labile lipids, but the ionization efficiency for less polar lipids was poor. Both methods were successfully applied to the direct analysis of lipids from pharmaceutical and food products. Although DESI and DAPPI provide efficient analysis of lipids, the multiple and largely unpredictable ionization reactions may set challenges for routine lipid analysis with these methods. Copyright © 2012 John Wiley & Sons, Ltd.     

Lipidomic-based investigation into the therapeutic effects of polyene phosphatidylcholine and Babao Dan on rats with non-alcoholic fatty liver disease

In recent years, with the improvement of people’s living standards, non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in the world. In this paper, the metabolic disorders in Sprague Dawley (SD) rats were induced by a choline-deficient, l -amino acid–defined (CDAA) diet. The therapeutic effects of polyene phosphatidylcholine (PPC) and Babao Dan (BBD) on NAFLD were observed. Lipidomic analysis was performed using ultra-high-performance liquid chromatography-Orbitrap MS, and data analysis and lipid identification were performed using the software LipidSearch. Both PPC and BBD can reduce lipid accumulation in the liver and improve abnormal biochemical indicators in rats, including reduction of triglycerides, total cholesterol, alanine transaminase and aspartate transaminase in serum. In addition, lipids in rat serum were systematically analyzed by lipidomics. The lipidomic results showed that the most obvious lipids with abnormal metabolism in CDAA diet–induced rats were glycerides (triglycerides and diacylglycerols), phospholipids and cholesterol esters. Both BBD and PPC partly reversed the disturbance to lipids induced by the CDAA diet. PPC may be more effective than BBD in alleviating NAFLD because it has a better effect on inhibiting the abnormal accumulation of lipids and reducing the inflammatory reaction in the body.     

A lipidomic platform establishment for structural identification of skin ceramides with non-hydroxyacyl chains

The stratum corneum (SC) is the outermost layer of skin that functions as a barrier and protects against environmental influences and transepidermal water loss. Its unique morphology consists of keratin-enriched corneocytes embedded in a distinctive mixture of lipids containing mainly ceramides, free fatty acids, and cholesterol. Ceramides are sphingolipids consisting of sphingoid bases, which are linked to fatty acids by an amide bond. Typical sphingoid bases in the skin are composed of dihydrosphingosine (dS), sphingosine (S), phytosphingosine (P), and 6-hydroxysphingosine (H), and the fatty acid acyl chains are composed of non-hydroxy fatty acid (N), α-hydroxy fatty acid (A), ω-hydroxy fatty acid (O), and esterified ω-hydroxy fatty acid (E). The 16 ceramide classes include several combinations of sphingoid bases and fatty acid acyl chains. Among them, N-type ceramides are the most abundant in the SC. Mass spectrometry (MS)/MS analysis of N-type ceramides using chip-based direct infusion nanoelectrospray-ion trap mass spectrometry generated the characteristic fragmentation pattern of both acyl and sphingoid units, which could be applied to structural identification of ceramides. Based on the MS/MS fragmentation patterns of N-type ceramides, comprehensive fragmentation schemes were proposed. In addition, mass fragmentation patterns, which are specific to the sphingoid backbone of N-type ceramides, were found in higher m/z regions of tandem mass spectra. These characteristic and general fragmentation patterns were used to identify N-type ceramides in human SC. Based on established MS/MS fragmentation patterns of N-type ceramides, 52 ceramides (including different classes of NS, NdS, NP, and NH) were identified in human SC. The MS/MS fragmentation patterns of N-type ceramides were characterized by interpreting their product ion scan mass spectra. This information may be used to identify N-type ceramides in the SC of human, rat, and mouse skin.     

Mapping and sequencing of cardiolipins from Geobacillus stearothermophilus NRS 2004/3a by positive and negative ion nanoESI-QTOF-MS and MS/MS

In the course of systematic studies on surface layer (S-layer) glycoproteins of bacilli, the chloroform/methanol extract from whole cells of Geobacillus stearothermophilus NRS 2004/3a has been submitted to MS analysis. Glucosylated cardiolipins were found as minor components of the total lipid and phospholipid mixture by de novo identification. After purification of the crude extract using a combined column chromatography/2D TLC protocol, structural investigations of components in the lipid fraction by high resolution ESI-QTOF MS analysis provided evidence about homologous molecules attributable to the cardiolipin species containing a glycosylated backbone, and about a diversity of ester-linked fatty acid substituents. In comparative studies by positive and negative ion nanoESI-QTOF-CID-MS, maps of cardiolipin molecular ions were obtained, followed by MS/MS of the most abundant species, to provide structural details of D-glucopyranosylcardiolipin and the fatty acid substituent patterns. Experiments of the parent ion scan type revealed the presence of fatty acid moieties as isobaric combinations, represented in single molecular ion species.     

Mass spectrometric evidence for the modification of small molecules in a cobalt‐60‐irradiated rodent diet

The purpose of this study was to investigate the effect of radiation on the content of animal diet constituents using global metabolomics. Aqueous methanolic extracts of control and cobalt‐60‐irradiated Teklad 7001 diets were comprehensively analyzed using nano‐liquid chromatography–MS/MS. Among the over 2000 ions revealed by XCMS followed by data preprocessing, 94 positive and 143 negative metabolite ions had greater than 1.5‐fold changes and p‐values <0.01. Use of MetaboAnalyst statistical software demonstrated complete separation of the irradiated and non‐radiated diets in unsupervised principal components analysis and supervised partial least squares discriminant analysis. Irradiation led to an increase in the content of phytochemicals such as glucosinolates and oxidized lipids in the diet. Twenty‐eight metabolites that were significantly changed in the irradiated samples were putatively identified at the level of molecular formulae by MS/MS. MS/MS^ALL analysis of chloroform–methanol extracts of the irradiated diet showed increased levels of a number of unique linoleic acid‐derived branched fatty acid esters of hydroxy fatty acids. These data imply that gamma irradiation of animal diets causes chemical changes to dietary components, which in turn may influence the risk of mammary cancer. Copyright © 2017 John Wiley & Sons, Ltd.     

The Structure of a Novel Neutral Lipid A from the Lipopolysaccharide of Bradyrhizobium elkanii Containing Three Mannose Units in the Backbone

The chemical structure of the lipid A of the lipopolysaccharide (LPS) from Bradyrhizobium elkanii USDA 76 (a member of the group of slow‐growing rhizobia) has been established. It differed considerably from lipids A of other Gram‐negative bacteria, in that it completely lacks negatively charged groups (phosphate or uronic acid residues); the glucosamine (GlcpN) disaccharide backbone is replaced by one consisting of 2,3‐dideoxy‐2,3‐diamino‐D ‐glucopyranose (GlcpN3N) and it contains two long‐chain fatty acids, which is unusual among rhizobia. The GlcpN3N disaccharide was further substituted by three D ‐mannopyranose (D ‐Manp) residues, together forming a pentasaccharide. To establish the structural details of this molecule, 1D and 2D NMR spectroscopy, chemical composition analyses and high‐resolution mass spectrometry methods (electrospray ionisation Fourier‐transform ion cyclotron resonance mass spectrometry (ESI FT‐ICR MS) and tandem mass spectrometry (MS/MS)) were applied. By using 1D and 2D NMR spectroscopy experiments, it was confirmed that one D ‐Manp was linked to C‐1 of the reducing GlcpN3N and an α‐(1→6)‐linked D ‐Manp disaccharide was located at C‐4′ of the non‐reducing GlcpN3N (α‐linkage). Fatty acid analysis identified 12:0(3‐OH) and 14:0(3‐OH), which were amide‐linked to GlcpN3N. Other lipid A constituents were long (ω‐1)‐hydroxylated fatty acids with 26–33 carbon atoms, as well as their oxo forms (28:0(27‐oxo) and 30:0(29‐oxo)). The 28:0(27‐OH) was the most abundant acyl residue. As confirmed by high‐resolution mass spectrometry techniques, these long‐chain fatty acids created two acyloxyacyl residues with the 3‐hydroxy fatty acids. Thus, lipid A from B. elkanii comprised six acyl residues. It was also shown that one of the acyloxyacyl residues could be further acylated by 3‐hydroxybutyric acid (linked to the (ω‐1)‐hydroxy group).     

Fatty Acids Profiling and Biomarker Identification in Snow Alga Chlamydomonas Nivalis by NaCl Stress Using GC/MS and Multivariate Statistical Analysis

The snow alga Chlamydomonas nivalis is a model species of microalgae for the investigation of cell response mechanism and adaptation ability in natural habitats in polar regions and similar extreme environments. The alteration of fatty acids in cellular lipids is known to play a vital role for cell survival and reproduction under various stress conditions. In the present work, an integrated approach of gas chromatography/mass spectrometry (GC/MS) coupled with multivariate statistical analysis was developed to investigate the fatty acid profiles and identify the biomarkers in response to NaCl stress. The data of fatty acid profiles between the control and NaCl-stress group was classified by orthogonal projection on latent structure discriminant analysis (OPLS-DA) and hierarchical cluster analysis (HCA); six of fatty acids (C16:0, C16:3, C18:0, C18:1, C18:2, and C18:3) were identified as biomarkers. These biomarkers showed a regulatory role by decreasing the degree of lipid unsaturation (DLU), providing an expected function in reducing membrane fluidity and permeability for enhancing the tolerance to higher salinity. This is the first report to demonstrate the fatty acid biomarkers in microalgae as the physiological regulators corresponding to the response and adaptation to NaCl stress based on an integrated approach at the lipidomic level.     

Changes in lipid composition of apple surface layer during long-term storage in controlled atmosphere

Apples are the most frequently consumed fruit and about 90 % of apple production is stored. Fatty acids and lipids are important constituents of plant cells. Disturbances in the lipid composition of fruit may lead to various stress processes, resulting in some storage disorders. This work is focused on an analysis of surface lipids of different varieties of apples stored in a normal atmosphere and a modified atmosphere with ultra-low oxygen content, for 4 months and 6 months. The major fatty acids in apple surface layers are palmitic acid, stearic acid, linoleic acid, and oleic acid. During the 6-months storage period, a variety-specific decrease in the total fatty acids content and an increase in saturation degree was observed in all the varieties tested, when compared with the 4-months storage. The greatest differences in saturation degree were observed in the Golden Delicious variety, in which the highest content of unsaturated fatty acids was also found. Microbial contamination of apple surfaces increased gradually over the storage process. Higher fungi levels were found in apples stored in the regular atmosphere than in the modified atmosphere, which can be attributed to changes observed in the total lipid content and saturation degree of the surface fatty acids and also to the sensitivity of microorganisms to the oxygen content in the storage room.     

Localization of cyclopropyl groups and alkenes within glycerophospholipids using gas-phase ion/ion chemistry

Shotgun lipid analysis using electrospray ionization tandem mass spectrometry (ESI-MS/MS) is a common approach for the identification and characterization of glycerophohspholipids GPs. ESI-MS/MS, with the aid of collision-induced dissociation (CID), enables the characterization of GP species at the headgroup and fatty acyl sum compositional levels. However, important structural features that are often present, such as carbon–carbon double bond(s) and cyclopropane ring(s), can be difficult to determine. Here, we report the use of gas-phase charge inversion reactions that, in combination with CID, allow for more detailed structural elucidation of GPs. CID of a singly deprotonated GP, [GP − H]⁻, generates FA anions, [FA − H]⁻. The fatty acid anions can then react with doubly charged cationic magnesium tris-phenanthroline complex, [Mg(Phen)₃]²⁺, to form charge inverted complex cations of the form [FA − H + MgPhen₂]⁺. CID of the complex generates product ion spectral patterns that allow for the identification of carbon–carbon double bond position(s) as well as the sites of cyclopropyl position(s) in unsaturated lipids. This approach to determining both double bond and cyclopropane positions is demonstrated with GPs for the first time using standards and is applied to lipids extracted from Escherichia coli.     

Non‐polar lipids characterization of Quinoa (Chenopodium quinoa) seed by comprehensive two‐dimensional gas chromatography with flame ionization/mass spectrometry detection and non‐aqueous reversed‐phase liquid chromatography with atmospheric pressure chemical ionization mass spectrometry detection

A chemical characterization of major lipid components, namely, triacylglycerols, fatty acids and the unsaponifiable fraction, in a Quinoa seed lipids sample is reported. To tackle such a task, non‐aqueous reversed‐phase high‐performance liquid chromatography with mass spectrometry detection was employed. The latter was interfaced with atmospheric pressure chemical ionization for the analysis of triacylglycerols. The main triacylglycerols (>10%) were represented by OLP, OOL and OLL (P = palmitoyl, O = oleoyl, L = linoleoyl); the latter was present in the oil sample at the highest percentage (18.1%). Furthermore, fatty acid methyl esters were evaluated by gas chromatography with flame ionization detection. 89% of the total fatty acids was represented by unsaturated fatty acid methyl esters with the greatest percentage represented by linoleic and oleic acids accounting for approximately 48 and 28%, respectively. An extensive characterization of the unsaponifiable fraction of Quinoa seed lipids was performed for the first time, by using comprehensive two‐dimensional gas chromatography with dual mass spectrometry/flame ionization detection. Overall, 66 compounds of the unsaponifiable fraction were tentatively identified, many constituents of which (particularly sterols) were confirmed by using gas chromatography with high‐resolution time‐of‐flight mass spectrometry.     

Mass spectrometry and inflammation—MS methods to study oxidation and enzyme-induced changes of phospholipids

Many diseases such as arthritis or atherosclerosis are accompanied by inflammatory processes. Inflammation is characterized by the infiltration of cells such as neutrophilic granulocytes and (a) the release of phospholipases [particularly phospholipase A₂ (PLA₂)] and (b) the generation of reactive oxygen as well as nitrogen species (ROS and RNS). While PLA₂ leads to defined lyso products (lacking one acyl residue), lipid oxidation is characterized by much more complex product patterns, including lipid peroxides, aldehydes (by double bond cleavage), and many others. Nevertheless, oxidation processes are highly important under in vivo conditions because molecules with regulatory functions are generated by the oxidation of lipids and/or free fatty acids. Therefore, lipid oxidation products as well as lysolipids are increasingly assumed to represent important disease (bio)markers. Consequently, there is also increasing interest in methods to characterize these products qualitatively and quantitatively. Mass spectrometry (MS) seems to be the method of choice to study (phospho)lipids changed under inflammatory conditions: nowadays, soft ionization MS methods are regularly used to study oxidative lipid modifications because of their high sensitivities and the tremendous mass resolutions that are achievable by using modern mass spectrometers. However, experimental care is required to be able to detect all relevant products. Although electrospray ionization (ESI) MS is so far most popular, applications of matrix-assisted laser desorption and ionization (MALDI) MS are continuously increasing. This review aims to summarize the so far available data on MS analyses of oxidized lipids as well as lysolipids. In addition to model systems, special attention will be paid to the monitoring of oxidized lipids and lysolipids under in vivo conditions. It is the aim of this review to provide a critical survey of the advantages and drawbacks of the different MS methods, with the focus on MALDI and ESI.

Regiospecific analysis of neutral ether lipids by liquid chromatography/electrospray ionization/single quadrupole mass spectrometry: validation with synthetic compounds.

A reversed-phase high-performance liquid chromatography (HPLC) method with on-line electrospray ionization/collision-induced dissociation/mass spectrometry (ESI/CID/MS) is presented for the regiospecific analysis of synthetic reference compounds of neutral ether lipids. The reference compounds were characterized by chromatographic retention times, full mass spectra, and fragmentation patterns as an aid to clarify the regiospecificity of ether lipids from natural sources. The results clearly show that single quadrupole mass spectroscopic analysis may elucidate the regiospecific structure of neutral ether lipids. Ether lipid reference compounds were characterized by five to six major ions in the positive ion mode. The 1-O-alkyl-sn-glycerols were analyzed as the diacetoyl derivative, and showed the [M - acetoyl](+) ion as an important diagnostic ion. The diagnostic ions of directly analyzed 1-O-alkyl-2-acyl-sn-glycerols and 1-O-alkyl-3-acyl-sn-glycerols were the [M - alkyl](+), [M + H - H(2)O](+) and [M + H](+) ions. Regiospecific characterization of the fatty acid position was evident from the relative ion intensities, as the sn-2 species had relatively high [M + H](+) ion intensities compared with [M + H - H(2)O](+), whereas the reverse situation characterized the sn-3 species. Furthermore, corresponding sn-2 and sn-3 species were separated by the chromatographic system. However, loss of water was promoted as fatty acid unsaturation was raised, which may complicate interpretation of the mass spectra. The diagnostic ions of directly analyzed 1-O-alkyl-2,3-diacyl-sn-glycerols were the [M - alkyl](+), [M - sn-2-acyl](+) and [M - sn-3-acyl](+) ions. Regiospecific characterization of the fatty acid identity and position was evident from the relative ion intensities, as fragmentation of the sn-2 fatty acids was preferred to the sn-3 fatty acids; however, loss of fatty acids was also promoted by higher degrees of unsaturation. Therefore, both structural and positional effects of the fatty acids affect the spectra of the neutral ether lipids. Fragmentation patterns and optimal capillary exit voltages are suggested for each neutral ether lipid class. The present study demonstrates that reversed-phase HPLC and positive ion ESI/CID/MS provide direct and unambiguous information about the configuration and identity of molecular species in neutral 1-O-alkyl-sn-glycerol classes.