Comparative molecular dynamics study of ether- and ester-linked phospholipid bilayers

The lipid membranes found in archaea have high bilayer stability and low permeability. The molecular structure of their constituent lipids is characterized by ether-linked, branched hydrophobic chains, whereas the conventional lipids obtained from eukaryotic or eubacterial sources have ester linked straight chains. In order to elucidate the influence of the ether linkage, instead of an ester one, on the physical properties of the lipid bilayers, we have carried out comparative 10 ns molecular dynamics simulations of diphytanyl phosphatidylcholine (ether-DPhPC) and diphytanoyl phosphatidylcholine (ester-DPhPC) bilayers in water, respectively. We analyze bilayer structures, hydration of the lipids, membrane dipole potentials, and free energy profiles of water and oxygen across the bilayers. We observe that the membrane dipole potential for the ether-DPhPC bilayer, which arises mainly from the ether linkage, is about half of that of the ester-DPhPC. The calculated free energy barrier for a water molecule in the ether-DPhPC bilayer system is slightly higher than that in the ester-DPhPC counterpart, which is in accord with experimental data.     

Fast atom bombardment mass spectrometry as a rapid means of screening mixtures of ether-linked polar lipids from extremely halophilic archaebacteria for the presence of novel chemical structures

Fast atom bombardment mass spectrometry was used to analyse intact polar ether lipids present at microgram levels in crude lipid mixtures extracted from Halobacterium halobium, Natronococcus occultus and Halobacterium marismortui. Negative-ion spectra showed the intact deprotonated lipid molecules and in some instances their sodium salts. The simplicity of the mass spectra permits the rapid screening of polar lipid mixtures for the presence of novel lipids. Additional structural information of ions with selected masses was obtained after collisionally induced decomposition.     

Rapid discrimination of archaeal tetraether lipid cores by liquid chromatography-tandem mass spectrometry

Atmospheric pressure chemical ionization liquid chromatography-tandem mass spectrometry (APCI LC-MS/MS) of tetraether lipid cores of archaeal origin reveals distinct dissociation pathways for three classes of core lipid extracted from Methanobacter thermautotrophicus. Within these classes, two isobaric tetraether lipids, one a scarcely reported lipid constituent of M. thermautotrophicus and the other an artefact formed during extraction from cultured cells, were identified and distinguished via their MS(2) spectra. APCI LC-MS/MS discriminates different tetraether core lipid types and isobaric species and reveals the mass of the constituent biphytanyl chains within the tetraether cores, albeit without full elucidation of their structures. Furthermore, the method allows direct estimation of the relative proportions of tetraether core lipids from chromatographic peak area measurement, allowing rapid profiling of these compounds in microbiological and environmental extracts.     

Molecular dynamics simulation of an archaeal lipid bilayer with sodium chloride

We have performed molecular dynamics simulations of a bilayer formed by the synthetic archaeal lipid, diphytanyl phosphatidylcholine, in NaCl electrolyte solution at four different concentrations (0-4 M) to investigate how structural and dynamic properties of the model archaeal membrane are changed due to the ionic strength of the solution. The archaeal lipid bilayer shows minor changes in their physical properties, indicating the unusual high stability of the membrane against salt, though small reductions of molecular area and lateral diffusion of the lipid are detected at the highest electrolyte concentration of 4 M. Sodium ions penetrate to the ether-rich region, where the ions are likely bound to the ether oxygen in the sn-1 chain rather than to that in the sn-2 chain. The observed salt bridges among two or three neighboring lipids account for the small reduction in the molecular area. The bound ions together with the counter (chloride) ions give rise to a diffusive electric double layer; as a result, the membrane dipole potential is slightly increased with increasing NaCl concentration.     

Determination of amphetamine in rat brain by in vivo microdialysis and ion-pairing liquid chromatography with electrospray tandem mass spectrometry

An ion-pairing liquid chromatography/electrospray tandem mass spectrometry (LC/ES-MS/MS) method with in vivo microdialysis for the determination of amphetamine in rat brain has been developed. A microdialysis probe was surgically implanted into the striatum of the rat and artificial cerebrospinal fluid (aCSF) was used as the perfusion medium. Samples were collected and then analyzed off-line by LC/ES-MS/MS. A reversed-phase C18 column was employed for LC separation and MS/MS was utilized for detection. Trifluoroacetic acid (TFA) was added to the mobile phase (acetonitrile/water) as an ion-pairing reagent. Detection was by ES-MS/MS directly, and no post-column addition of organic modifier was needed. Dual linear ranges were determined from 0.1-0.5 microg/mL and 0.005-0.1 microg/mL, respectively. The detection limit, based on a signal-to-noise ratio of 3, was 0.001 microg/mL (5 nM). Good precision and accuracy were obtained. The applicability of this newly developed method was demonstrated by continuous monitoring of amphetamine concentrations in rat brain. Amphetamine reached a maximum concentration of 0.086 +/- 0.017 microg/mL over 20-40 min after a single 3.0 mg/kg intraperitoneal administration.     

Analysis of intact ladderane phospholipids, originating from viable anammox bacteria, using RP-LC-ESI-MS

Since the discovery of the anaerobic ammonium oxidizing (anammox) bacteria, many attempts have been made in order to identify these environmentally important bacteria in natural environments. Anammox bacteria contain a unique class of lipids, called ladderane lipids and here we present a novel method to detect viable anammox bacteria in sediments and waste water treatment plants based on the use of a ladderane lipid biomarker. Intact ladderane phosphatidylcholine (PC) lipids are analyzed using reversed-phase liquid chromatography–electrospray ionization–mass spectrometry. Following extraction from the complex sediment matrix, reversed-phase LC is used to separate ladderane PC lipids based on their tail group hydrophobicity as well as their ether or ester link to the glycerol backbone in the sn-2 position. We investigate the presence of intact ladderane lipids in natural sediments displaying anammox activity and illustrate the use of a specific intact membrane forming PC lipid as a biomarker for viable anammox bacterial cells. The presented method can be used to elucidate the whereabouts of viable anammox bacteria, subsequently enabling an estimation of anammox activity. This will greatly increase the knowledge of anammox bacteria and their importance in the global nitrogen cycle.     

Characterization of polysorbate 85, a nonionic surfactant,by liquid chromatography vs. ion mobility separation coupled with tandem mass spectrometry

Liquid chromatography (LC) and ion mobility (IM) separation have been coupled with mass spectrometry (MS) and tandem mass spectrometry (MS²) to characterize a commercially important nonionic surfactant, polysorbate 85. The constituents of this amphiphilic blend contained a sorbitan or isosorbide core that was chain extended with poly(ethylene oxide) (PEO) and partially esterified at the PEO termini with oleic acid or, to a lesser extent, other fatty acids. Using interactive LC in reverse-phase mode, the oligomers of the surfactant were separated according to their hydrophobicity/hydrophilicity balance. On the other hand, IM spectrometry dispersed the surfactant oligomers by their charge and collision cross section (i.e. size/shape). With either separation method, an increased number of fatty ester groups and/or lack of the polar sorbitan (or isosorbide) core led to higher retention/drift times, enabling the separation of isobaric species or species with superimposed isotope patterns, so that their ester content could be conclusively identified by MS². LC–MS and IM–MS permitted the detection of several byproducts besides the major PEO-sorbitan oleate oligomers. LC–MS provides the separation resolution needed for quantitative determination of the degree of esterification. IM–MS, which minimizes analysis time and solvent use, is ideally suitable for a fast, qualitative survey of samples differing in their minor constituents or impurities.     

Identification of bupropion urinary metabolites by liquid chromatography/mass spectrometry

Human urinary metabolism of the antidepressant bupropion was studied using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). A total of 20 metabolites were detected and identified. The phase I metabolism included formation of morpholinohydroxybupropion, threo- and erythrohydrobupropion, aromatic hydroxylation, butyl group hydroxylation with ketone hydrogenation and dihydroxylation. These metabolites were detected either as the free form or as glucuronide and/or sulphate conjugates. In addition also m-chlorohippuric acid was detected. Of the phase I metabolites, a dihydroxylation to the aromatic ring and to the methyl group in the middle of the substrate molecule was reported here for the first time, as well as eight of the glucuronide conjugates (to hydroxy, dihydroxy, hydroxy and hydrogenation metabolites) and three of the sulphate conjugates (to aromatic hydroxy and hydroxy and hydrogenation metabolites).     

Corniculoside,a New Biosidic Ester Secoiridoid from Halenia corniculata

Chemical screening of the secondary metabolites from Halenia corniculata L. (CORNAZ ), by LC/UV,LC/TSP-MS (thermospray), and LC/ES-MS (electrospray) was used for the targeted isolation of corniculoside ( 1 ), a new biosidic ester secoiridoid. The structure was established as 7-β-[(E)-4′-O-(β-D -glucopyranosyl)caffeoyloxy]-sweroside by 1D- and 2D-NMR, LC/UV, LC/MS, and FAB-MS data, in combination with chemical reactions.     

Simultaneous quantitative analyses of indole and oxindole alkaloids of Uncaria Hook in rat plasma and brain after oral administration of the traditional Japanese medicine Yokukansan using high‐performance liquid chromatography with tandem mass spectrometry

Uncaria Hook (UH) alkaloids are involved in the beneficial effects of Yokukansan. However, the pharmacokinetics of UH alkaloids after oral administration of Yokukansan has not yet been sufficiently investigated. Therefore, we developed and validated a sensitive and specific high‐performance liquid chromatography with tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantitation of seven UH alkaloids (corynoxeine, isocorynoxeine, rhynchophylline, isorhynchophylline, hirsutine, hirsuteine and geissoschizine methyl ether) in rat plasma and brain. After protein precipitation with acetonitrile, chromatographic separation was performed using an Ascentis Express RP‐amide column, with gradient elution with 0.2% formic acid and acetonitrile at 0.3 mL/min. All analytes in the plasma and brain showed good linearity over a wide concentration range (r > 0.995). Intra‐day and inter‐day variations of each constituent were 8.6 and 8.0% or less in the plasma, and 14.9 and 15.0% or less in the brain, respectively. The validated LC/MS/MS method was applied in the pharmacokinetic studies of UH alkaloids after oral administration of Yokukansan to rats. In the plasma, rhynchophylline, hirsutine, hirsuteine and geissoschizine methyl ether were detected, but only geissoschizine methyl ether was detected in the brain. These results suggest that geissoschizine methyl ether is an important constituent of the pharmacological effects of Yokukansan. Copyright © 2013 John Wiley & Sons, Ltd.     

Spectral characterisation of eight glycerolipids and their detection in natural samples using time‐of‐flight secondary ion mass spectrometry

In recent years, time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) with cluster ion sources has opened new perspectives for the analysis of lipid biomarkers in geobiology and organic geochemistry. However, published ToF‐SIMS reference spectra of relevant compounds are still sparse, and the influence of the chemical environment (matrix) on the ionisation of molecules and their fragmentation is still not well explored. This study presents ToF‐SIMS spectra of eight glycerolipids as common target compounds in biomarker studies, namely ester‐ and ether‐bound phosphatidylethanolamine, ester‐ and ether‐bound phosphatidylcholine, ester‐bound phosphatidylglycerol, ester‐ and ether‐bound diglycerides and archaeol, obtained with a Bi cluster ion source. For all of these compounds, the spectra obtained in positive and negative analytical modes showed characteristic fragments that could clearly be assigned to e.g. molecular ions, functional groups and alkyl chains. By comparison with the reference spectra, it was possible to track some of these lipids in a pre‐characterised organic extract and in cryosections of microbial mats. The results highlight the potential of ToF‐SIMS for the laterally resolved analysis of organic biomarkers in environmental materials. The identification of the target compounds, however, may be hampered by matrix effects (e.g. adduct formation) and often require careful consideration of all spectral features and taking advantage of the molecular imaging capability of ToF‐SIMS. Copyright © 2009 John Wiley & Sons, Ltd.     

Two‐dimensional LC‐MS/MS to enhance ceramide and phosphatidylcholine species profiling in mouse liver

A two‐dimensional (2D) hydrophilic interaction liquid chromatography (HILIC) and reverse‐phase (RP) liquid chromatography (LC) system coupled with triple‐quadrupole mass spectrometry (MS) was developed to comprehensively profile ceramides and phosphatidylcholine in extracted biological samples. Briefly, the 2D HILIC‐RPLC system used a silica HILIC column operated in the first dimension to distinguish the lipid classes and a BEH C₁₈ column operated in the second dimension to separate the lipid species of the same class. The regression linearity of each lipid was satisfactory in both systems; however, the absolute matrix effect factor was reduced in 2D LC‐MS/MS system. Limits of detection of 2D LC‐MS/MS system were 2‐ to 3‐fold lower compared with one‐dimensional RPLC‐MS/MS. The recovery from the sample ranged from 84.5 to 110%. To summarize, the developed method was proven to be accurate and producible, as relative standard deviations remained <20% at three spiked levels. The efficiency of this newly developed system was applied to measure changes of lipids in the liver of mice after naphthalene treatment. Orthogonal projection to latent structures‐discriminant analysis discriminated the lipids from control and the treatment group. We concluded that 2D LC‐MS/MS is a promising method to assist lipidomic studies of complex biological samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of phenothiazines in human body fluids by solid-phase microextraction and liquid chromatography/tandem mass spectrometry

Eleven phenothiazine derivatives with heavy side-chains were found to be extractable from human whole blood and urine samples by solid-phase microextraction (SPME) with a polyacrylate-coated fiber. The fiber was then injected into the desorption chamber of an SPME-liquid chromatography (LC) interface for LC/tandem mass spectrometry (MS/MS) with positive ion electrospray (ES) ionization. All compounds formed base peaks due to [M + 1](+) ions by LC/ES-MS/MS. By use of LC/ES-MS/MS, the product ions produced from each [M + 1](+) ion showed base peaks due to side-chain liberation. Selected reaction monitoring (SRM) and selected ion monitoring (SIM) were compared for the detection of the 11 phenothiazine derivatives from human whole blood and urine. SRM showed much higher sensitivity than SIM for both types of sample. Therefore, a detailed procedure for the detection of drugs by SRM with SPME-LC/MS/MS was established and carefully validated. The extraction efficiencies of the 11 phenothiazine derivatives spiked into whole blood and urine were 0. 0002-0.12 and 2.6-39.8%, respectively. The regression equations for the 11 phenothiazine derivatives showed excellent linearity with detection limits of 0.2-200 ng ml(-1) for whole blood and 4-22 pg ml(-1) for urine. The intra- and inter-day precisions for whole blood and urine samples were not greater than 15.1%. The data obtained after oral administration of perazine or flupentixol to a male subject are presented.     

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction, followed by high-speed liquid chromatography/mass spectrometry, for the determination of a basic drug in human plasma

A simplified protein precipitation/mixed-mode cation-exchange solid-phase extraction (PPT/SPE) procedure has been investigated. A mixture of acetonitrile and methanol along with formic acid was used to precipitate plasma proteins prior to selectively extracting the basic drug. After vortexing and centrifugation, the supernatants were directly loaded onto an unconditioned Oasis MCX microElution 96-well extraction plate, where the protonated drug was retained on the negatively charged sorbent while interfering neutral lipids, steroids or other endogenous materials were washed away. Normal wash steps were deemed unnecessary and not used before sample elution. The sample extracts were analyzed under both conventional and high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) conditions to examine the feasibility of the PPT/SPE procedure for human plasma sample clean-up. For the conventional LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 50 mm column with gradient elution (k' = 5.5). The mobile phase contained 0.1% formic acid in water and 0.1% formic acid in acetonitrile. For the high-speed LC/MS/MS method, chromatographic separation was achieved on a C18, 2.1 x 10 mm guard column with gradient elution (k' = 2.2, Rt = 0.26 min). The mobile phase contained 0.1% formic acid in water and 0.001% trifluoroacetic acid in acetonitrile. Detection for both conventional and high-speed LC/MS/MS methods was by positive ion electrospray tandem mass spectrometry on a ThermoElectron Finnigan TSQ Quantum Ultra, where enhanced resolution (RP 2000; 0.2 amu) was used for high-speed LC/MS/MS. The standard curve, ranging from 0.5 to 100 ng/mL, was fitted to a 1/x weighted quadratic regression model.This combined PPT/SPE procedure effectively eliminated time-consuming sorbent conditioning and wash steps, which are essential for a conventional mixed-mode SPE procedure, but retained the advantages of both PPT (removal of plasma proteins) and mixed-mode SPE (analyte selectivity). The validation results demonstrated that this PPT/SPE procedure was well suited for both conventional and high-speed LC/MS/MS analyses. In comparison with a conventional mixed-mode SPE procedure, the simplified PPT/SPE process provided comparable sample extract purity. This simple sample clean-up procedure can be applied to other basic compounds with minor modifications of PPT solvents.     

Detection of new propofol metabolites in human urine using gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry techniques

Using hyphenated analytical techniques, gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), a study on minor propofol metabolites in human urine was conducted. These techniques allowed identification of two new phase I metabolites (2-(omega-propanol)-6-isopropylphenol and 2-(omega-propanol)-6-isopropyl-1,4-quinol). In addition, their four corresponding conjugates (three glucuronides and one sulphate) were detected. Thus in human urine at least eight conjugate metabolites are produced, derived from four different aglycones (propofol; 2, 6-diisopropyl-1,4-quinol; 2-(omega-propanol)-6-isopropylphenol and 2-(omega-propanol)-6-isopropyl-1,4-quinol).     

The direct determination of double bond positions in lipid mixtures by liquid chromatography/in-line ozonolysis/mass spectrometry

The direct determination of double bond positions in unsaturated lipids using in-line ozonolysis-mass spectrometry (O₃-MS) is described. In this experiment, ozone penetrates through the semi-permeable Teflon AF-2400 tubing containing a flow of a solution of fatty acid methyl esters (FAME). Unsaturated FAME are thus oxidized by the ozone and cleaved at the double bond positions. The ozonolysis products then flow directly into the atmospheric pressure photoionization (APPI) source of a mass spectrometer for analysis. Aldehyde products retaining the methyl ester group are indicative of the double bond positions in unsaturated FAME. For the first time, O₃-MS is able to couple directly to high performance liquid chromatography (HPLC), making the double bond localization in lipid mixtures possible. The application of LC/O₃-MS has been demonstrated for a fat sample from bovine adipose tissue. A total of 9 unsaturated FAME including 6 positional isomers were identified unambiguously, without comparison to standards. The in-line ozonolysis reaction apparatus is applicable to most mass spectrometers without instrumental modification; it is also directly compatible with various LC columns. The LC/O₃-MS method described here is thus a practical, versatile and easy to use new approach to the direct determination of double bond positions in lipids, even in complex mixtures.     

Direct Analysis of Triacylglycerols from Crude Lipid Mixtures by Gold Nanoparticle-Assisted Laser Desorption/Ionization Mass Spectrometry

Triacylglycerols (TAGs), essential energy storage lipids, are easily detected by conventional MALDI MS when occurring on their own. However, their signals are easily overwhelmed by other lipids, mainly phosphatidylcholines (PCs) and, therefore, require purification. In order to profile TAGs from crude lipid mixtures without prefractionation, we investigated alternative matrixes that can suppress phospholipid ion signals and enhance cationization of TAGs. We found that an aqueous solution of citrate-capped gold nanoparticles (AuNPs) with a diameter of 12 nm is a superior matrix for the laser desorption/ionization mass spectrometry (LDI MS) of TAGs in crude lipid mixtures. The AuNP matrix effectively suppressed other lipid signals such as phospholipids and also provided 100 times lower detection limit for TAGs than 2,5-dihydroxybenzoic acid (DHB), the best conventional MALDI matrix for TAGs. The AuNP-assisted LDI MS enabled us to obtain detailed TAG profiles including minor species directly from crude beef lipid extracts without phospholipid interference. In addition, we could detect TAGs at a trace level from a total brain lipid extract.

Identification and relative quantification of fatty acids in Escherichia coli membranes by gas chromatography/mass spectrometry

The lipids that are essential to the functioning of the bacterial membrane exist in hundreds of different forms. The reasons for this diversity are far from clear but are presumably related to the roles of these lipids in both facilitating enzymic activities and generating proteolipid domains. A full understanding of bacterial physiology therefore requires characterization of lipids in different strains in a variety of environmental conditions. This characterization then becomes the basis for lipidomics, the lipid aspect of the growing field of metabolomics. To exploit the power of derivatization chemistry and of gas chromatography/mass spectrometry (GC/MS) and tandem mass spectrometry (MS/MS) for metabolomics studies, we report here the development of various GC/MS electron ionization (EI) and negative and positive chemical ionization (CI) methods for the identification and, for the first time, the relative quantification of fatty acids present in extracts from membranes of a laboratory strain of Escherichia coli. They consist of seven saturated fatty acids (C10:0, C12:0, C14:0, C15:0, C16:0, C17:0 and C18:0) and six unsaturated fatty acids (C16:1, cyC17:0 plus two isomers of C18:1, C18:2 and cyC19:0).     

Chromatographic and mass spectral studies of perfluorooctanesulfonate and three perfluorooctanesulfonamides

The chromatographic and mass spectral characteristics of perfluorooctanesulfonate (PFOS) and three nitrogen-substituted perfluorooctanesulfonamides have been obtained. A methyl/phenyl mixed-phase fused-silica capillary column was used for gas chromatographic (GC) analyses, while a C18 reversed-phase microbore column was used for liquid chromatographic (LC) analyses. Mass (MS) and tandem mass (MS/MS) spectra were generated using electron ionization (EI), argon CE, methane positive and negative ion CI, and ES ionization modes. EI spectra of the amides showed ions characteristic of both the fluorinated hydrocarbon and the sulfonamide portion of the molecules. The fragmentation pathway was studied using hydrogen/deuterium exchange, and was thought to involve a cyclic intermediate ion. Formation of molecular ions by CE and protonated molecule ions by CI to obtain molecular weight information was only partially successful. Negative ion ES-MS spectra provided intense [M-H]- anions for the amides, and an [M-K]- anion for PFOS from which molecular weight information could be obtained, while ES-MS/MS produced product ions that could be used to detect the presence of these compounds in biological or environmental samples.     

Reducing glycerophosphocholine lipid matrix interference effects in biological fluid assays by using high-turbulence liquid chromatography

Matrix interferences can severely affect quantitative assays of biological samples when electrospray ionization (ESI) is employed with liquid chromatography/tandem mass spectrometry (LC/MS/MS). A major source of matrix interferences for plasma sample analyses is the presence of glycerophosphocholine (GPCho) lipids. The efficiency of online high-turbulence liquid chromatography (HTLC) extraction for eliminating these lipids is evaluated and the interfering effects of endogenous lipids on human plasma assays are measured for pharmaceutical compounds having a wide variety of chemical properties. It is found that GPCho lipids, represented by 16:0, 18:1 and 18:0 LPC (lysophosphatidylcholine) and 16:0-18:2 PC, cause variations for hydrophobic compound analyses even when optimal online HTLC extraction conditions are employed. The efficiency for lipid removal depends on the organic content of the transfer solvent, but turbulent flow loading has no significant effect.