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.     

Triacylglycerol profiling of microalgae strains for biofuel feedstock by liquid chromatography-high-resolution mass spectrometry

Biofuels from photosynthetic microalgae are quickly gaining interest as a viable carbon-neutral energy source. Typically, characterization of algal feedstock involves breaking down triacylglycerols (TAG) and other intact lipids, followed by derivatization of the fatty acids to fatty acid methyl esters prior to analysis by gas chromatography (GC). However, knowledge of the intact lipid profile could offer significant advantages for discovery stage biofuel research such as the selection of an algal strain or the optimization of growth and extraction conditions. Herein, lipid extracts from microalgae were directly analyzed by ultra-high pressure liquid chromatography–mass spectrometry (UHPLC-MS) using a benchtop Orbitrap mass spectrometer. Phospholipids, glycolipids, and TAGs were analyzed in the same chromatographic run, using a combination of accurate mass and diagnostic fragment ions for identification. Using this approach, greater than 100 unique TAGs were identified over the six algal strains studied and TAG profiles were obtained to assess their potential for biofuel applications. Under the growth conditions employed, Botryococcus braunii and Scenedesmus obliquus yielded the most comprehensive TAG profile with a high abundance of TAGs containing oleic acid.     

Distinct hepatic lipid profile of hypertriglyceridemic mice determined by easy ambient sonic-spray ionization mass spectrometry

Easy ambient sonic-spray ionization mass spectrometry (EASI-MS) was used to interrogate the hepatic lipid profiles of hypertriglyceridemic and control normotriglyceridemic mice. The analyses of ex vivo complex lipid mixtures were made directly with EASI-MS without accompanying separation steps. Intense ions for phosphatidylcholines and triacylglycerols were observed in the positive ion mode whereas the spectra in the negative ion mode provided profiles of phosphatidylethanolamines and phosphatidylinositol. EASI-MS was coupled to high-performance thin-layer chromatography for analysis of free fatty acids. Fourier transform–ion cyclotron resonance–mass spectrometry was also employed to confirm the identity of the detected lipids. We demonstrated higher incorporation of oleic acid in phosphatidylcholine and triacylglycerol composition, higher relative abundance of arachidonic acid containing phosphatidylinositol, and overall distinct free fatty acid profile in the livers of genetic hypertriglyceridemic mice. We propose that these alterations in liver lipid composition are related to the higher tissue and body metabolic rates described in these hypertriglyceridemic mice.     

Desorption electrospray ionization imaging mass spectrometry of lipids in rat spinal cord

Imaging mass spectrometry allows for the direct investigation of tissue samples to identify specific biological compounds and determine their spatial distributions. Desorption electrospray ionization (DESI) mass spectrometry has been used for the imaging and analysis of rat spinal cord cross sections. Glycerophospholipids and sphingolipids, as well as fatty acids, were detected in both the negative and positive ion modes and identified through tandem mass spectrometry (MS/MS) product ion scans using collision-induced dissociation and accurate mass measurements. Differences in the relative abundances of lipids and free fatty acids were present between white and gray matter areas in both the negative and positive ion modes. DESI-MS images of the corresponding ions allow the determination of their spatial distributions within a cross section of the rat spinal cord, by scanning the DESI probe across the entire sample surface. Glycerophospholipids and sphingolipids were mostly detected in the white matter, while the free fatty acids were present in the gray matter. These results show parallels with reported distributions of lipids in studies of rat brain. This suggests that the spatial intensity distribution reflects relative concentration differences of the lipid and fatty acid compounds in the spinal cord tissue. The “butterfly” shape of the gray matter in the spinal cord cross section was resolved in the corresponding ion images, indicating that a lateral resolution of better than 200 μm was achieved. The selected ion images of lipids are directly correlated with anatomic features on the spinal cord corresponding to the white and the gray matter.     

气相色谱/质谱法分析孔石莼中的脂肪酸

建立了孔石莼脂肪酸的气相色谱/质谱(GC/MS)测定方法。使用Folch法提取了孔石莼中的总脂,经过2 mol/L HCl-甲醇溶液的甲酯化处理后,采用GC/MS法对其脂肪酸组成进行了分离分析,同时结合有机质谱学规律,分别对饱和脂肪酸甲酯、单不饱和脂肪酸甲酯和多不饱和脂肪酸甲酯的裂解规律和质谱特征进行了分析归纳。通过质谱数据库检索和标准品对照,鉴定出孔石莼中的24种脂肪酸,其中9,12,15-十八碳三烯酸、4,7,10,13-十六碳四烯酸和6,9,2,15-十八碳四烯酸3种主要多不饱和脂肪酸占总脂肪酸含量的45.14%。通过对孔石莼中脂肪酸的分析,表明特征离子在脂肪酸甲酯尤其是多不饱和脂肪酸甲酯的定性方面具有很好的应用价值。     

Formation of lithiated adducts of glycerophosphocholine lipids facilitates their identification by electrospray ionization tandem mass spectrometry

Electrospray ionization (ESI) tandem mass spectrometry (MS) has simplified analysis of phospholipid mixtures, and, in negative ion mode, permits structural identification of picomole amounts of phospholipid species. Collisionally activated dissociation (CAD) of phospholipid anions yields negative ion tandem mass spectra that contain fragment ions representing the fatty acid substituents as carboxylate anions. Glycerophosphocholine (GPC) lipids contain a quaternary nitrogen moiety and more readily form cationic adducts than anionic species, and positive ion tandem mass spectra of protonated GPC species contain no abundant ions that identify fatty acid substituents. We report here that lithiated adducts of GPC species are readily formed by adding lithium hydroxide to the solution in which phospholipid mixtures are infused into the ESI source. CAD of [MLi⁺] ions of GPC species yields tandem mass spectra that contain prominent ions representing losses of the fatty acid substituents. These ions and their relative abundances can be used to assign the identities and positions of the fatty acid substituents of GPC species. Tandem mass spectrometric scans monitoring neutral losses of the head-group or of fatty acid substituents from lithiated adducts can be used to identify GPC species in tissue phospholipid mixtures. Similar scans monitoring parents of specific product ions can also be used to identify the fatty acid substituents of GPC species, and this facilitates identification of distinct isobaric contributors to ions observed in the ESI/MS total ion current.     

Atmospheric pressure laser desorption/ionization of plant metabolites and plant tissue using colloidal graphite

Colloidal graphite is a promising matrix for atmospheric pressure laser desorption/ionization mass spectrometry. Intact [M+H]⁺ and [M–H]^? ions are readily produced from a wide range of small molecule plant metabolites, particularly anthocyanins, fatty acids, lipids, glycerides, and ceramides. Compared with a more traditional organic acid matrix, colloidal graphite provides more efficient ionization for small hydrophobic molecules and has a much cleaner background spectrum, especially in negative ion mode. Some important metabolites, e.g., fatty acids and glycosylated flavonoids, can be observed from Arabidopsis thaliana leaf and flower petal tissues in situ. Copyright © 2010 John Wiley & Sons, Ltd.     

Regioisomer characterization of triacylglycerols by non‐aqueous reversed‐phase liquid chromatography/electrospray ionization mass spectrometry using silver nitrate as a postcolumn reagent

Triacylglycerols (TAGs) provide a challenge for mass spectrometry (MS) analysis because of their complexity. In particular, for dietary, nutritional and metabolic purposes, the positional placement of fatty acids on the glycerol backbone of TAGs is a crucial aspect. To solve this problem, we have investigated the TAGs' fragmentation patterns using an ion trap mass spectrometer. A series of pure regioisomeric pairs of TAGs (POP/PPO, POO/OPO and OSO/SOO) were cationized by Ag⁺ after their separation by non‐aqueous reversed‐phase liquid chromatography (NARP‐LC) before MS to improve MS sensitivity. Electrospray ionization–MS (ESI‐MS) conditions were optimized in order to produce characteristic [M + Ag + AgNO₃]⁺ ions from each TAG, which were then fragmented to produce MS/MS spectra and then fragmented further to produce up to MS⁵ spectra. The observation of ions produced by LC‐MS⁵ of on‐line Ag⁺‐cationized TAG provided unambiguous information on the fatty acid distribution on the glycerol backbone. These strategies of MS to MS⁵ experiments were applied to identify components and to determine the regiospecificity of TAG within a complex mixture of lipids in natural oils. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of deprotonated acids with silicon nanoparticle-assisted laser desorption/ ionization mass spectrometry

Chemically modified silicon nanoparticles were applied for the laser desorption/negative ionization of small acids. A series of substituted sulfonic acids and fatty acids was studied. Compared to desorption ionization on porous silicon (DIOS) and other matrix-less laser desorption/ionization techniques, silicon nanoparticle-assisted laser desorption/ionization (SPALDI) mass spectrometry allows for the analysis of acids in the negative ion mode without the observation of multimers or cation adducts. Using SPALDI, detection limits of many acids reached levels down to 50 pmol/μl. SPALDI of fatty acids with unmodified silicon nanoparticles was compared to SPALDI using the fluoroalkyl silylated silicon powder, with the unmodified particles showing better sensitivity for fatty acids, but with more low-mass background due to impurities and surfactants in the untreated silicon powder. The fatty acids exhibited a size-dependent response in both SPALDI and unmodified SPALDI, showing a signal intensity increase with the chain length of the fatty acids (C12-C18), leveling off at chain lengths of C18-C22. The size effect may be due to the crystallization of long chain fatty acids on the silicon. This hypothesis was further explored and supported by SPALDI of several, similar sized, unsaturated fatty acids with various crystallinities. Fatty acids in milk lipids and tick nymph samples were directly detected and their concentration ratios were determined by SPALDI mass spectrometry without complicated and time-consuming purification and esterification required in the traditional analysis of fatty acids by gas chromatography (GC). These results suggest that SPALDI mass spectrometry has the potential application in fast screening for small acids in crude samples with minimal sample preparation.     

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.     

Electron transfer dissociation of doubly sodiated glycerophosphocholine lipids

The ability to generate gaseous doubly charged cations of glycerophosphocholine (GPC) lipids via electrospray ionization has made possible the evaluation of electron-transfer dissociation (ETD) for their structural characterization. Doubly sodiated GPC cations have been reacted with azobenzene radical anions in a linear ion trap mass spectrometer. The ion/ion reactions proceed through sodium transfer, electron-transfer, and complex formation. Electron-transfer reactions are shown to give rise to cleavage at each ester linkage with the subsequent loss of a neutral quaternary nitrogen moiety. Electron-transfer without dissociation produces [M + 2Na](+.) radical cations, which undergo collision-induced dissociation (CID) to give products that arise from bond cleavage of each fatty acid chain. The CID of the complex ions yields products similar to those produced directly from the electron-transfer reactions of doubly sodiated GPC, although with different relative abundances. These findings indicate that the analysis of GPC lipids by ETD in conjunction with CID can provide some structural information, such as the number of carbons, degree of unsaturation for each fatty acid substituent, and the positions of the fatty acid substituents; some information about the location of the double bonds may be present in low intensity CID product ions.     

Fast atom bombardment mass spectrometry; a promising tool for structural studies

Fast atom bombardment mass spectrometry (FABMS) is a powerful new structural tool. Ionization is effected by bombarding a glycerol matrix with neon or argon atoms of 5–10 KeV energy. Positive and negative secondary ions are sputtered from the surface and can be detected for inorganic ion clusters to mass 25 800 and biologically important compounds to mass 5 700. FABMS is especially valuable in determining the sequence of amino acids in polypeptides.     

The use of formic acid in carrier gas. Rapid method for identification and determination of phytanic acid by gas chromatography-mass spectrometry-chemical ionization

A rapid gas chromatographic method to determine phytanic acid in plasma from Refsum's disease is described. After a brief alkaline hydrolysis of lipids, the biological sample is directly injected into a glass pre-column; an acid carrier gas (formic acid in nitrogen) is used to displace the long-chain fatty acids from their sodium salts and from their binding to proteins. Formic acid introduced through the column may also be used as a reagent gas for chemical ionization in combined gas chromatography--mass spectrometry; fatty acids (C14 to C18:2 and phytanic acid) are easily identified by their M + 1 (base peak) and M - 17 peaks. The described procedure is also suitable for studying normal fatty acids from plasma lipids.     

Charge-remote fragmentation: an account of research on mechanisms and applications

Charge-remote fragmentation is one class of decomposition reactions of gas-phase ions. Their discovery and applications had to await the development of soft ionization, particularly fast atom bombardment, and tandem mass spectrometry. The decompositions are particularly informative and allow functional groups to be identified and located in fatty acids, surfactants, steroids, complex lipids, and related materials. These are difficult structure assignments to make by other mass-spectrometry methods or by nuclear magnetic resonance and other spectroscopic techniques. Thus, charge-remote fragmentation fills an important need in structural chemistry. The mechanisms of charge-remote fragmentation underpin their structural utility, and they are still a matter of some debate. In this account, we discuss the discovery of charge-remote fragmentation in our laboratory. Further, we describe our efforts to understand their mechanism and to exploit their high information content in structure determinations of fatty acid and related materials.     

Die massenspektrometrische Fragmentierung von Neopentylpolyolestern. 1—Pentaerythrittetrafettsäureester

The fragmentation of the homologous fatty acid tetraesters of pentaerythritol (C-2 to C-14) upon electron impact was investigated. The main fragment ions are [M? RCOO]⁺ and [M? RCOOH]⁺, for which cyclic acetal structures are postulated. Subsequent fragmentation was elucidated by ‘direct analysis of daughter ion’ (DADI) measurements and high resolution measurements. Esters of branched fatty acids can be distinguished from esters of n-fatty acids by characteristic ions. Isomeric esters of n-fatty acids cannot be separated by gas chromatography but identification is also possible by mass spectrometry.     

一些含锗有机酸的快原子轰击质谱

本文报道了一系列含锗有机酸在甘油中的快原子轰击质谱（ＦＡＢＭＳ）及共在间硝基苄醇（ＮＢＡ）中的正、负离子ＦＡＢＭＳ。这些含锗有机酸在不同的底物中的ＦＡＢＭＳ显示了不同的特征。负离子谱提供了分子量信息，正、负离子谱可为这类化合物的结构鉴定提供互相补充的信息。讨论了底物和取代基对ＦＡＢＭＳ的影响，正、负离子的产生机制及离子的亚稳分解途径。     

Quantification of low molecular weight fatty acids in cave drip water and speleothems using HPLC-ESI-IT/MS — development and validation of a selective method

This study presents a novel method for the analysis of low molecular weight (LMW) fatty acids in cave drip water and speleothems. The method development included optimization of sample preparation procedures, e.g., blank reduction, solid phase extraction, concentration of extracts as well as liquid chromatography coupled to electrospray ion-trap mass spectrometry (HPLC-ESI-IT/MS) measurement parameters. Retention times for five analytes (lauric acid, myristic acid, palmitic acid, stearic acid and arachidic acid) were between 5 and 13.5 min. Spiking experiments were performed to accomplish external calibrations which ranged from 12.5 to 75 ng per spiked water sample. The correlation coefficient ranged from 0.9558 to 0.9989. Inter-batch precision, expressed as the relative standard deviation of three replicates, was <7 %. Limits of detection ranged from 0.77 to 55.97 ng for the diverse analytes; obtained recoveries varied from 30 to 103 %. For a first application, cave drip water and stalagmite samples from Herbstlabyrinth-Adventhöhle cave system were analyzed. Concentrations ranged from 38.37 to 9,982.54 ng L^?1 for water samples and 2.52 to 1,344.96 ng g^?1 for the stalagmite. Thereby, the different fatty acids showed a distinctive variation. Whereas shorter-chained fatty acids exhibited similarities, arachidic acid showed opposite trends. Diverse correlations were found, which could improve the understanding of different organic sources of the lipids transported by drip water and preserved in speleothems. This new method provides a more selective extraction process, particularly adjusted to LMW fatty acids and therefore reduces the required sample size. Furthermore, it is applicable to stalagmite as well as cave drip water samples.     

Fast-atom-bombardment and tandem mass spectrometry for determining structures of fatty acids as their picolinyl ester derivatives

Picolinyl ester derivatives of common fatty acids can be readily desorbed by fast atom bombardment (FAB) as positive ions and then collisionally activated. Collisionally activated spectra of the (M + H)⁺ ions of the derivatives reveal that structurally informative remote-charge-site fragmentations occur. The presence of substitutents such as double bond, branch points, cyclopropane rings, hydroxy groups, and epoxy rings interrupts the fragmentation process in such a way that the substituent can be identified and its location on the alkyl chain can be determined. This method is also applicable to the picolinyl esters of short-chain fatty acids and to the analysis of mixtures of fatty acid derivatives. The approach is advantageous becasue the epicolinyl ester derivatives are also amenable to gas chromatography/mass spectrometry (GC/MS). Therefore, the FAB-MS/MS approach developed here is complementary to GC/MS.     

Complementary analysis of lipids in whole bacteria cells by thermally assisted hydrolysis and methylation-GC and MALDI-MS combined with on-probe sample pretreatment

The molecular structure of lipids in whole bacteria cells was characterized in detail by using two different and complementarily direct analyses; thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with on-probe sample pretreatment. First, THM-GC in the presence of tetramethylammonium hydroxide (TMAH) was applied to compositional analysis of the fatty acid components of lipids in whole bacterial cells. On the resulting chromatograms, a series of fatty acid components in bacterial lipids were clearly observed as resolved peaks of their methyl esters. The fatty acid compositions determined on the basis of the peak intensities were in good agreement with those obtained by the conventional technique involving solvent extraction of the lipids in the samples. Furthermore, MALDI-MS combined with the on-probe sample treatment, using 2,5-dihydroxybenzoic acid (DHB) and sodium iodide (NaI) as matrix and cationization reagents, respectively, was used to detect intact phospholipids directly from whole bacterial cells. The MALDI spectra thus obtained showed an array of ions generated from bacterial phospholipids, such as phosphatidylethanolamines (PEs) and phosphatidylglycerols (PGs). Finally, the bacterial lipids were comprehensively characterized in terms of the acyl groups and the molecular structures by taking both of the results obtained by THM-GC and MALDI-MS into consideration.     

海蜇不同部位脂肪酸的组成研究

报道了黄海海域成熟海蜇不同部位脂肪酸的研究结果。利用GC-MS技术测定海蜇皮、海蜇头、海蜇生殖腺的脂肪酸组成。结果表明：海蜇含有30多种脂肪酸,3个部位的脂肪酸组成差别不大;海蜇中含有丰富的多不饱和脂肪酸,含量在36．2％～38．7％之间,其中二十二碳六烯酸(DHA)、二十碳四烯酸(AA)和二十碳五烯酸(EPA)含量较高。