Quantitative profiling of phosphatidylcholine and phosphatidylethanolamine in a steatosis/fibrosis model of rat liver by nanoflow liquid chromatography/tandem mass spectrometry

Quantitative analysis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was carried out using a steatosis/fibrosis model of rat livers, which were induced by a chronic administration of carbon tetrachloride (CCl(4)). Intact phospholipid mixtures from each liver sample (from rats fed with 0.5 mL CCl(4)/kg three times a week for 30, 60, and 90 days) were analyzed by nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (nLC/ESI/MS/MS), and identifications of 37 PC and 19 PE species were made by collision-induced dissociation. The quantitative analysis utilized a multiple standard addition method with an internal standard, and the relationship between the MS peak intensities of different PC species and their carbon chain length was included for calibration. It was found that the total amount of PC and PE species decreased significantly with administration of CCl(4). While concentrations of most PC and PE species decreased to a great extent, three PEs and seven PCs were up-regulated more than twofold.     

Photosensitized oxidation of phosphatidylethanolamines monitored by electrospray tandem mass spectrometry

Photodynamic therapy combines visible light and a photosensitizer (PS) in the presence of molecular oxygen to generate reactive oxygen species able to modify biological structures such as phospholipids. Phosphatidylethanolamines (PEs), being major phospholipid constituents of mammalian cells and membranes of Gram‐negative bacteria, are potential targets of photosensitization. In this work, the oxidative modifications induced by white light in combination with cationic porphyrins (Tri‐Py⁺‐Me‐PF and Tetra‐Py⁺‐Me) were evaluated on PE standards. Electrospray ionization mass spectrometry (ESI‐MS) and tandem mass spectrometry (ESI‐MS/MS) were used to identify and characterize the oxidative modifications induced in PEs (POPE: PE 16:0/18:1, PLPE: PE 16:0/18:2, PAPE: PE 16:0/20:4). Photo‐oxidation products of POPE, PLPE and PAPE as hydroxy, hydroperoxy and keteno derivatives and products due to oxidation in ethanolamine polar head were identified. Hydroperoxy‐PEs were found to be the major photo‐oxidation products. Quantification of hydroperoxides (PE‐OOH) allowed differentiating the potential effect in photodamage of the two porphyrins. The highest amounts of PE‐OOH were notorious in the presence of Tri‐Py⁺‐Me‐PF, a highly efficient PS against bacteria. The identification of these modifications in PEs is an important key point in the understanding cell damage processes underlying photodynamic therapy approaches. Copyright © 2013 John Wiley & Sons, Ltd.     

Determination of the fatty acyl profiles of phosphatidylethanolamines by tandem mass spectrometry of sodium adducts

Phosphatidylethanolamines (PEs) are one of the major constituents of cellular membranes, and, along with other phospholipid classes, have an essential role in the physiology of cells. Profiling of phospholipids in biological samples is currently done using mass spectrometry (MS). In this work we describe the MS fragmentation of sodium adducts of 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and 2-linoleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine (PLPE). This study was performed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) using three different instruments and also by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). All MS/MS spectra show product ions related to the polar head fragmentation and product ions related to the loss of acyl chains. In ESI-MS/MS spectra, the product ions [M+Na-R1COOH-43]+ and [M+Na-R2COOH-43]+ show different relative abundance, as well as [M+Na-R1COOH]+ and [M+Na-R2COOH]+ product ions, allowing identification of both fatty acyl residues of PEs, and their specific location. MALDI-MS/MS shows the same product ions reported before and other ions generated by charge-remote fragmentation of the C3-C4 bond (gamma-cleavage) of fatty acyl residues combined with loss of 163 Da. These fragment ions, [M+Na-(R2-C2H3)-163]+ and [M+Na-(R1-C2H3)-163]+, show different relative abundances, and the product ion formed by the gamma-cleavage of sn-2 is the most abundant. Overall, differences noted that are important for identification and location of fatty acyl residues in the glycerol backbone are: relative abundance between the product ions [M+Na-R1COOH-43]+ > [M+Na-R2COOH-43]+ in ESI-MS/MS spectra; and relative abundance between the product ions [M+Na-(R2-C2H3)-163]+ > [M+Na-(R1-C2H3)-163]+ in MALDI-MS/MS spectra.     

A shotgun tandem mass spectrometric analysis of phospholipids with normal-phase and/or reverse-phase liquid chromatography/electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is used in lipidomics studies. The present research established a top-down liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) shotgun analysis method for phospholipids (PLs) using a normal-phase column or a C30 reverse-phase column with the data-dependent MS/MS scanning mode. A normal-phase column can separate most of the major different classes of PLs. By using LC/ESI-MS/MS with a normal-phase column, approximately 50 molecular species were identified in a PL mixture from rat liver. When the reverse-phase column was used, the PLs could be separated depending on their hydrophobicity, essentially the length of their fatty acyl chains and the number of unsaturated bonds in them. The LC/ESI-MS/MS method using a C30 reverse-phase column was applied to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixtures as test samples. Molecular species with the same molecular mass but with different pairs of fatty acyl chains were separately identified. As a result, about 60 PC and 50 PE species were identified. PLs from rat liver were subjected to LC/ESI-MS/MS using the C30 reverse-phase column and about 110 molecular species were identified. Off-line two-dimensional LC/ESI-MS/MS with the normal-phase and C30 reverse-phase columns allowed more accurate identification of molecular species by using one-dimensional C30 reverse-phase LC/ESI-MS/MS analysis of the collected fractions.     

The new matrix 4-Chloro-α-cyanocinnamic acid allows the detection of phosphatidylethanolamine chloramines by MALDI-TOF mass spectrometry

Phosphatidylethanolamines (PEs) are abundant lipid constituents of the cellular membrane. The amino group of PEs exhibits high reactivity with hypochlorous acid that is generated under inflammatory conditions in vivo. The analysis of the resulting PE mono- and dichloramines is of significant interest since these species represent important mediators of lipid peroxidation. We have shown in a previous communication that mass spectrometric detection of PE chloramines is only possible with ESI MS, whereas MALDI-TOF MS fails to detect these products if standard matrices are used.     

Rapid identification of glycerophospholipids from RAW264.7 cells by UPLC/ESI ‐QTOF‐MS

The study aims to develop a rapid, sensitive ultra‐performance liquid chromatography coupled with an electrospray ionization quadruple time‐of‐flight tandem mass spectrometry (UPLC/Q‐TOF‐MS) analytical method for identifying glycerophospholipids (GPLs) from RAW264.7 cells. A total of 78 GPLs including 22 phosphatidylethanolamines (PEs), 49 phosphatidylcholines (PCs), four phosphatidylglycerols, one phosphatidylinositol and two unknown GPLs were identified. PC (14:0/16:1), PC (14:0/16:0), PE (0:0/20:3), PE (22:5/0:0) and PE (22:3/0:0) were identified for the first time. The UPLC/Q‐TOF‐MS method is suitable for targeting analysis of GPLs from RAW264.7 cells, which allows us to find out new GPLs compositions related to inflammatory diseases and to explain their pharmacological roles in inflammatory process. Copyright © 2014 John Wiley & Sons, Ltd.     

Photooxidation of glycated and non‐glycated phosphatidylethanolamines monitored by mass spectrometry

Phosphatidylethanolamines (PE) are one of the major components of cells membranes, namely in skin and in retina, that are continuously exposed to solar UV radiation being major targets of photooxidation damage. In addition, due to the presence of the free amine group, PE can also undergo glycation, in hyperglycemic conditions which may increase the susceptibility to oxidation. The aim of this study is to develop a model, based on mass spectrometry (MS) analysis, to identify photooxidative degradation of selected PE (POPE: PE 16:0/18:1, PLPE: PE 16:0/18:2, PAPE: PE 16:0/20:4) and glycated PEs due to UV irradiation. Photooxidation products were analysed by electrospray ionization MS (ESI‐MS) and tandem MS (ESI‐MS/MS) in positive and negative mode. Emphasis is placed in the influence of glycation in the generation of distinct photooxidation products. ESI‐MS spectra of PE after UV photo‐irradiation showed mainly hydroperoxy derivatives, due to oxidation of unsaturated fatty acyl chains. Glycated PE gave rise to several new photooxidation products formed due to oxidative cleavages of the glucose moiety, namely between C1 and C2, C2 and C3, and C5 and C6 of this sugar unit. These new products were identified by ESI‐MS/MS in positive mode showing distinct neutral loss depending on the different structure of the polar head group. These new identified advanced glycated photooxidation products may have a deleterious role in the etiology of diabetic retinopathy and in diabetic retinal microvascular complications. Copyright © 2013 John Wiley & Sons, Ltd.     

Palladium-catalyzed oxyarylation of olefins using silver carbonate as the base. Probing the mechanism by electrospray ionization mass spectrometry

The Pd(OAc)₂-catalyzed oxyarylation of electron-rich (8 and 12) and electron-poor (10) olefins by ortho-iodophenols (3a-d) was studied using Ag₂CO₃ as the base, in acetone, and in the presence and absence of PPh₃. The corresponding adducts of oxyarylation were obtained in moderate yields. The reaction mechanism was examined by electrospray ionization mass spectrometry (ESI-MS). Cationic arylpalladium intermediate (14), formed by the oxidative insertion of Pd(0) into 3a, and the cationic palladacycles (15), obtained by reaction of 14 with olefins 8 and 12, were intercepted by ESI-MS and characterized by ESI-MS/MS.     

带羟乙基侧臂三脚架多胺Cu(Ⅱ)配合物切割肌红蛋白所得片断的质谱指认

采用电喷雾质谱和串联质谱以及聚丙烯酰胺凝胶电泳技术研究了[CuL(H₂O)](BF₄)₂(L为2-[二(2-氨乙酸)氨基]乙醇)与马心肌红蛋白的键合作用和水解切割。聚丙烯酰胺凝胶电泳研究显示在中性及60 ℃条件下，切割效率与[CuL(H₂O)]²⁺的浓度和温育时间密切相关。电喷雾质谱和串联质谱分析显示，[CuL(H₂O)]²⁺通过与肌红蛋白的氨基酸His36，His93，His116和Arg139侧链的结合，并在羟乙基侧臂的促进下，选择性地水解了肽键Phe33-Thr34，Gln91-Ser92，Ala94-Thr95，His116-Ser117和Asn140-Asp141。     

Fluorene‐rich high performance polyesters: Synthesis and characterization of 9,9‐fluorenylidene and 2,7‐fluorenylene‐based polyesters with excellent optical property

Polyesters PEs containing high content of fluorene units in their backbones were synthesized from 9,9‐diarene‐substituted fluorene diols ( 1 ) and fluorene‐based diacid chlorides ( 2 ) by high temperature polycondensation at 185 °C in diphenyl ether. The molecular weights of the polyesters PE1‐PE5 were in a range of M_w 25,000–165,000. The polyesters displayed their high thermostability: the glass transition temperatures (T_g) by differential scanning calorimetry analysis ranged from 109 to 217 °C, while the 10% weight loss temperatures (T_d10) measured by thermogravimetric analysis were over 400 °C in nitrogen and 395 °C in air. The polyesters had good solubility in most common organic solvents such as chloroform and toluene and gave tough, transparent and flexible cast films. The transmittance of the films was over 80% in the wavelength range from 450 to 700 nm in any PEs . The PEs exhibited high refractive index values around 1.65, while they had very low degree of birefringence. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 2549–2556, 2008     

Static secondary ion mass spectrometry of organic plasma-deposited films created from stable isotope-labeled precursors. II. Mixtures of acetone and oxygen

A series of plasma-deposited films (PDFs), created by blending controlled ratios of acetone vapor and oxygen in the feed to the plasma reactor, were analyzed by static secondary ion mass spectrometry (SIMS). Examination of the quadrupole-based static SIMS fragmentation patterns of acetone–O₂ PDFs created from ¹³C-labeled acetone generally allowed the hydrocarbon secondary ions to be distinguished from oxygen-containing secondary ions. These results were compared with those obtained via high-mass resolution time-of-flight (TOF)-SIMS. The identified secondary ions were then assigned structural attributes, based on comparison of the static SIMS spectra of the acetone–O₂ PDFs with those of conventional hydrocarbon and oxygen-containing polymers. A preliminary investigation to unravel the mechanism of oxygen incorporation in the acetone–O₂ PDFs from the two plasma feed components was undertaken through the analysis of PDFs created from 1,2,3¹³C₃-acetone(¹⁶O) vapor and ¹⁸O₂ gas.     

Determination of seven benzoylphenylurea insecticides in processed fruit and vegetables using high-performance liquid chromatography/tandem mass spectrometry

A liquid chromatographic method with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been developed for the sensitive and selective determination of seven benzoylphenylurea insecticide residues (diflubenzuron, triflumuron, lufenuron, flufenoxuron, teflubenzuron, chlorfuazuron, hexaflumuron) in pear baby purée, concentrated lemon juice, and tomato pulp. The general sample extraction/partition method for our established multiresidue methods has been used. The entire procedure involves extraction of residues with acetone and partition into ethyl acetate/cyclohexane. Chromatographic determination was performed using a C18 column and isocratic elution. Fourteen MS/MS transitions of precursor ions were monitored (two for each pesticide) using negative ESI. The majority of mean recoveries at fortification levels of 0.002-0.020 and 0.020-0.200 mg/kg were in the range 77-102% with relative standard deviations between 2 and 10%. The excellent sensitivity and selectivity of this LC/MS/MS method allowed quantitation and identification at low levels in difficult matrices with a run time of 4 min.     

固相萃取净化及超高效液相色谱-串联质谱法测定橡胶制品中的13种N-亚硝胺

建立了固相萃取(SPE)净化、超高效液相色谱-串联质谱(UHPLC-MS/MS)测定橡胶制品中13种N-亚硝胺的方法。样品于密闭萃取瓶中于60 ℃下用甲醇超声萃取30 min,C₁₈固相萃取小柱对萃取液进行净化,经C₁₈色谱柱分离,最后用电喷雾正离子(ESI⁺)和多重反应监测模式(MRM)对13种N-亚硝胺进行定性、定量测定。实验中对样品前处理、色谱分离条件和质谱检测条件进行了优化。在优化的实验条件下,橡胶样品中添加N-亚硝基二甲胺(NDMA)与N-亚硝基-二乙基胺(NDEA)为500 μg/kg、其他组分均为50 μg/kg时,各组分的相对标准偏差(RSD,n=7)小于10%;在实际样品中的加标回收率为70.7%~117.0%;方法的检出限(LOD,以10倍标准偏差计)为0.5~500 μg/kg。方法可应用于橡胶制品中13种N-亚硝胺的测定。     

Electrospray ionization mass spectrometry fingerprinting of propolis

Crude ethanolic extracts of propolis, a natural resin, have been directly analysed using electrospray ionization mass (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) in the negative ion mode. European, North American and African samples have been analyzed, but emphasis has been given to Brazilian propolis which displays diverse and region-dependent chemical composition. ESI-MS provides characteristic fingerprint mass spectra, with propolis samples being divided into well-defined groups directly related to their geographical origins. Chemometric multivariate analysis statistically demonstrates the reliability of the ESI-MS fingerprinting method for propolis. On-line ESI-MS/MS tandem mass spectrometry of characteristic [M - H](-) ion markers provides an additional dimension of fingerprinting selectivity, while structurally characterizing the ESI-MS marker components of propolis. By comparison with standards, eight such markers have been identified: para-coumaric acid, 3-methoxy-4-hydroxycinnamaldehyde, 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran, 3-prenyl-4-hydroxycinnamic acid, chrysin, pinocembrin, 3,5-diprenyl-4-hydroxycinnamic acid and dicaffeoylquinic acid. The negative mode ESI-MS fingerprinting method is capable of discerning distinct composition patterns to typify, to screen the sample origin and to reveal characteristic details of the more polar and acidic chemical components of propolis samples from different regions of the world.     

Light and heavy dansyl reporter groups in food chemistry: amino acid assay in beverages

5‐Dimethylamino‐1‐sulfonyl naphthalene (DNS, commonly referred as dansyl) is a functionality, bearing well‐established properties in directing the fragmentation, by mass spectrometry (MS), of the corresponding ionized sulfonylated derivatives. This property is shared also by its labeled analogs. The use of d₀/d₆ DNS derivatives is now exploited in the application of the well‐established isotope dilution mass spectrometric approach in the assay of complex mixtures. A new method for the quantitation of amino acids (AAs) in beverages is therefore presented, which relies on liquid chromatographic separation of their N‐dansylated derivatives followed by comparative electrospray tandem MS/MS of the d₀/d₆ isobaric mixtures. Labeled and unlabeled DNS derivatives of the selected AAs are readily available by microwave‐assisted synthetic protocols. The novelty of the method is represented by the use of heavy and light DNS‐isotopologue providing suitable reporter groups. Multiple‐reaction monitoring has been applied in the assay of AAs in wine, pineapple juice and bergamot juice with good‐to‐excellent results as proved by both relative standard deviation, lower than 15%, and by the accuracy values in the range 90–110%. Copyright © 2012 John Wiley & Sons, Ltd.     

Isotopic fractionation during pretreatment for accelerator mass spetrometer measurement of (D<Subscript>3</Subscript>C)<Subscript>2</Subscript>O containing <Superscript>14</Superscript>C produced by nuclear reaction

A (D₃C)₂O (d₆-acetone) target was irradiated with semi-monoenergetic neutrons generated from ⁹Be(p,n)⁹B reaction with 20 MeV protons to convert ¹³C and oxygen nuclides in the target into ¹⁴C. With both liquid scintillation counting (LSC) and accelerator mass spectrometry (AMS) we measured the (D₃C)₂O (d₆-acetone) liquid targets, which were combustible and easy to afford CO₂ for the AMS measurements. The ¹⁴C yield measured by the LSC method turned out to be 80 times larger than that by the AMS method. This large discrepancy may be attributed to the loss of ¹⁴C atoms during the sample pretreatment in the AMS method such as combustion and cryogenic trapping of CO₂. It means that ¹⁴C newly produced by nuclear reactions can exist in various chemical forms, i.e., C₃D₆O, CO, CO₂, hydrocarbons, etc., and a simple sample pretreatment right after production can cause serious isotopic fractionation. Therefore, using the AMS method, extreme caution in sample pretreatment should be exercised when the ¹⁴C yield produced immediately by nuclear reaction is measured.     

L-羟脯氨酸寡肽混合物的高效液相色谱分离与质谱分析

研究了三氯氧磷辅助下L-羟脯氨酸的成肽反应,建立了采用反相高效液相色谱-质谱/质谱联用技术分离鉴定羟脯氨酸寡肽混合物的方法,优化了L-羟脯氨酸寡肽混合物的色谱分离条件。实验以YWG C8柱(10 μm,250 mm×10 mm)为分离柱,以乙腈-0.06%三氟乙酸水溶液(体积比为2∶98)为流动相进行等度洗脱,在正离子模式下对洗脱物进行了电喷雾电离串联质谱鉴定。结果显示,分离出的各组分分别为L-羟脯氨酸二肽、L-羟脯氨酸环二肽和L-羟脯氨酸三肽。     

Matrix-assisted laser desorption/ionization directed nano-electrospray ionization tandem mass spectrometric analysis for protein identification

In those cases where the information obtained by peptide mass fingerprinting or matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) is not sufficient for unambiguous protein identification, nano-electrospray ionization (nano-ESI) and/or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis must be performed. The sensitivity of nano-ESI/MS, however, is lower than that of MALDI-MS, especially at very low analyte concentrations and/or in the presence of contaminants, such as salt and detergents. Moreover, to perform ESI-MS/MS, the peptide masses of the precursor ions must be known. The approach described in this paper, MALDI-directed nano-ESI-MS/MS, makes use of information obtained from the more sensitive MALDI-MS experiments in order to direct subsequent nano-ESI-MS/MS experiments. Peptide molecular ions found in the MALDI-MS analysis are then selected, as their (+2) precursor ions, for nano-ESI-MS/MS sequencing, even though these ions cannot be detected in the ESI-MS spectra. This method, originally proposed by Tempst et al. (Anal. Chem. 2000, 72: 777-790), has been extended to provide better sensitivity and shorter analysis times; also, a comparison with liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been performed. These experiments, performed using quadrupole time-of-flight instruments equipped with commercially available nano-ESI sources, have allowed the unambiguous identification of in-gel digested proteins at levels below their ESI-MS detection limits, even in the presence of salts and detergents.     

Determination of Carbon‐based Engineered Nanoparticles in Marketed Fish by Microwave‐assisted Extraction and Liquid Chromatography‐atmospheric Pressure Photoionization‐tandem Mass Spectrometry

An optimized method for the determination of two major carbon‐based engineered nanoparticles (C₆₀ and C₇₀) in marketed fish samples is described. The method involves the use of microwave‐assisted extraction (MAE) coupled with liquid chromatography ‐ tandem mass spectrometry with atmospheric pressure photoionization (LC‐APPI‐MS/MS). Factors affecting the extraction efficiency of the analytes from fish samples were optimized by a central composite design method. The optimal extraction temperature and time for MAE were found to be 233 °C for 22 min, and the extraction solution was composed of toluene and acetone in a ratio of 4.64:1. The limits of quantitation (LOQs) were 0.1 and 0.05 ng/g for C₆₀ and C₇₀, respectively. The precision for these analytes at two spiked levels, as indicated by relative standard deviations (RSDs), were less than 10% for both intra‐ and inter‐day analysis. Accuracy, expressed as the mean extraction recovery, was between 85 and 98%. The method was further validated based on EU Commission Decision 2002/657/EC, including a decision limit (CCα) and detection capability (CCβ) for marketed fish samples.     

Ligand size distribution of phenanthroline-functionalized polyethylene glycol-iron(II) complexes determined by electrospray ionization mass spectrometry and computer simulation

The complex formation in solution, and the gas-phase dissociation of a phenanthrolineterminated poly(ethylene glycol) with Fe²⁺ ions were investigated. The size distribution of poly(ethylene glycol)-α-monomethyl-ω-5-[1,10]phenanthroline (mPEG_phen) was determined by electrospray ionization mass spectrometry (ESI-MS). Based on the measured ligand size distribution of mPEG_phen by ESI-MS, the 1:3 complex formation (Fe²⁺/mPEG_Phen) was computer-simulated as a pure random assembly process. The simulated distribution fits excellently to that of the complex Fe(mPEG_phen)₃²⁺ determined from the ESI-MS intensities. In addition, the collision-induced dissociation (CID) of the Fe(mPEG_phen)₃²⁺ complex was also studied by tandem mass spectrometry (ESI-MS/MS) and by computer simulation, as well. The ESI-MS/MS intensity distribution of the Fe(mPEG_phen)₂²⁺ formed from Fe(mPEG_phen)₃²⁺ by the loss of an mPEG_phen ligand under CID conditions fits quite well to the calculated one.