Formation of [M-H]- and [M-2H]2- ions in the electrospray ionization mass spectra of dicarboxylated polyethylene glycols

The negative-ion electrospray mass spectrometric behavior of dicarboxylated polyethylene glycols (CPEGCs) is discussed. Both [M-H](-) and [M-2H](2-) ions were observed. It was found that the ratio [M-2H](2-)/[M-H](-) is affected by oxyethylene chain length, solvent polarity, analyte concentration and applied cone voltage.     

Generation of alkoxide anions from a series of aliphatic diols and alcohols and their ion-molecule reactions with carbon dioxide in the gas phase

Alkoxide anions, [M-H](-) from a series of aliphatic diols and alcohols are generated in the source under negative ion electrospray ionisation conditions by cone-voltage fragmentation of the corresponding [M + F](-) ions. The collision-induced dissociation (CID) spectra of [M-H](-) ions consist of [M-H-2H](-) ions, in addition to the other characteristic fragment ions, and the relative abundance of [M-H-2H(-) ions among the series of diols varies as a function of chain length that could be explained based on their stabilities through intramolecular hydrogen bonding. The reactivity of alkoxide anions is studied through ion-molecule reactions with CO(2) in the collision cell of a triple quadrupole mass spectrometer. All the alkoxide anions reacted with CO(2) and formed corresponding carbonate anions, [M-H + CO(2)](-) ions. The reactivity of alkoxide anions within the series of diols also reflected the stability of their [M-H](-) ions.     

Comparison of the positive and negative ion electrospray mass spectra of some small peptides containing pyroglutamate

The positive ion electrospray mass spectra of [M+H](+) and the negative ion electrospray mass spectra of [M-H](-) ions of selected pyroglutamate containing peptides both provide sequencing data. The negative ion spectra show the normal alpha and beta backbone cleavages in addition to delta and gamma backbone cleavages initiated by the side chains of Glu and Phe residues. For example, the [M-H](-) ion of pGlu Pro Gln Val Phe Val-NH(2) shows delta and gamma peaks at m/z 224 (delta, Gln3), 244 (gamma, Phe4), 451 (delta, Phe4), 471 (gamma, Gln3). Some of the negative ion spectra show unusual grandaughter peaks that originate by alpha and beta, or delta and gamma backbone cleavages of a beta(1) cleavage ion.     

Letter: Electrospray ionization mass spectral characteristics and fragmentation mechanisms of triterpenoids in Fomes officinalis

The electrospray ionization tandem mass spectrometric characteristics and fragmentation mechanisms of 12 triterpenoid compounds from Fomes officinalis (F. officinalis) and their analogs were investigated. The compounds could be classified into three types depending on their chemical structures. All of the compounds gave [M-H](-) and [2M-H](-) ions by electrospray negative ionization mode. In addition, the members of three isomeric groups of the analogs with the same elemental composition can be distinguished by tandem mass spectra of protonated molecules and of significant fragmention. The above fragmentations were reported for the first time and were implemented for the analysis of triterpenoids in F. officinalis.     

Structural analysis of selected characteristic flavones by electrospray tandem mass spectrometry.

Seven structure analogical flavonoid aglycones have been analyzed using electrospray ionization tandem mass spectrometry (ESI-MSn) in the negative-ion mode. The spectra obtained ESI-MSn allowed us to propose plausible schemes for their fragmentation mechanism. By analyzing the product ions spectra of deprotonated molecule ions [M-H](-), some neutral diagnostic losses and specific retro Diels-Alder fragments were obtained. By using all of these characteristic fragment ions we can specially differentiate the flavone isomer.     

Analytical application of acetate anion in negative electrospray ionization mass spectrometry for the analysis of triterpenoid saponins--ginsenosides

Ginsenosides containing different numbers of glycosyl groups can be easily differentiated based on the formation of characteristic ginsenoside-acetate adduct anions and deprotonated ginsenosides generated by electrospray ionization (ESI) of methanolic solutions of ginsenosides (M) and ammonium acetate (NH4OAc). Ginsenosides containing two glycosyl groups gave a characteristic mass spectral pattern consisting of [M+2OAc]2-, [M-H+OAc]2- and [M-2H]2- ions with m/z values differing by 30 Th, while this mass spectral pattern was not observed for ginsenosides containing one glycosyl group. Formation of [M+2OAc]2- was influenced by the chain length of glycosyl groups and was used to differentiate the ginsenosides containing different combinations of monosaccharide and disaccharide units in the glycosyl groups. Under identical collisional activation conditions, [M+OAc]-, [M-H+OAc]2- and [M+2OAc]2- underwent proton abstractions predominantly to generate [M-H]-, [M-2H]2- and [M-H+OAc]2- ions, respectively. The ion intensity ratios, I[M-H](-/I) [M+OAc]-, I[M-2H](2-/I) [M-H+2OAc]2- and I[M-H+OAc](2-/I) [M+OAc]2-, being sensitive to the structural differences of ginsenosides, could differentiate the isomeric ginsenosides, including (i) Rf, F11 and Rg1, (ii) Rd and Re, and (iii) Rb2 and Rc. Additionally, NH4OAc was found to enhance the sensitivity of detection of ginsenosides in the form of [M-H]- down to the femtomole level.     

Electrospray ionisation mass spectral studies on hydrolysed products of sulfur mustards

Off-site detection of the hydrolysed products of sulfur mustards in aqueous samples is an important task in the verification of Chemical Weapons Convention (CWC)-related chemicals. The hydrolysed products of sulfur mustards are studied under positive and negative electrospray ionisation (ESI) conditions using an additive with a view to detecting them at trace levels. In the presence of cations (Li(+), Na(+), K(+) and NH(4) (+)), the positive ion ESI mass spectra of all the compounds include the corresponding cationised species; however, only the [M+NH(4)](+) ions form [M+H](+) ions upon decomposition. The tandem mass (MS/MS) spectra of [M+H](+) ions from all the hydrolysed products of the sulfur mustard homologues were distinct and allowed these compounds to be characterised unambiguously. Similarly, the negative ion ESI mass spectra of all the compounds show prominent adducts with added anions (F(-), Cl(-), Br(-), and I(-)), but the [M-H](-) ion can only be generated by decomposition of an [M+F](-) ion. The MS/MS spectra of the [M-H](-) ions from all the compounds result in a common product ion at m/z 77. A precursor ion scan of m/z 77 is shown to be useful in the rapid screening of these compounds in aqueous samples at trace levels, even in the presence of complex masking agents, without the use of time-consuming sample preparation and chromatography steps. An MS/MS method developed to measure the detection limits of the hydrolysed products of sulfur mustards found these to be in the range of 10-500 ppb.     

Fragmentation reactions of phenoxide anions: deprotonated Dinoterb and related structures

Dinoterb (6-t-butyl-2,4-dinitrophenol), 1, Dinoseb (6-secbutyl-2,4-dinitrophenol), 2, TBP (2-t-butylphenol), 3, and DNP (2,4-dinitrophenol), 4, have been analyzed by electrospray ionization in the negative mode (ESI-N) - tandem mass spectrometry. Nominal laboratory collision energy was varied from zero to 60 eV during the experiments. Apparent fragmentation energies were estimated from a parametric fitting of the collision efficiency curves. In parallel, fragmentation mechanisms of the deprotonated molecules [M-H](-) were explored using quantum chemistry modeling at the B3LYP/6-31 + G(d,p) level. A major fragmentation of the [M-H](-) ions of Dinoterb and Dinoseb is elimination of an alcohol molecule. This reaction is shown to involve one oxygen atom originating from a nitro group rather than the phenoxide moiety. Eliminations of NO, C(4) and CH(2) = C(CH(3))(2), i.e. reactions involving significant rearrangements, constitute the major part of the other fragmentation pathways observed from [3-H](-) and [4-H](-) ions.     

Tools to discover anionic and nonionic polyfluorinated alkyl surfactants by liquid chromatography electrospray ionisation mass spectrometry

A tiered approach is proposed for the discovery of unknown anionic and nonionic polyfluorinated alkyl surfactants (PFASs) by reversed phase ultra high performance liquid chromatography (UHPLC)--negative electrospray ionisation--quadrupole time of flight mass spectrometry (UHPLC-ESI(-)-QTOF-MS). The chromatographic separation, ionisation and detection of PFASs mixtures, was achieved at high pH (pH=9.7) with NH(4)OH as additive. To distinguish PFASs from other chemicals we used the characteristic negative mass defects of PFASs, their specific losses of 20 Da (HF) and the presence of series of chromatographic peaks, belonging to homologues series with m/z of n×50 Da (CF(2)) or n×100 Da (CF(2)CF(2)). The elemental composition of the precursor ions were deducted from the accurate m/z values of the deprotonated molecules [M-H](-). In case of in-source fragmentation, the presence of dimers, e.g. [M(2)-H](-) and adduct ions such as [M-H+solvent](-) and [(M-H)(M-H+Na)(n)](-) were used to confirm the identity of the precursor ions. In relation to quantification of PFASs, we discuss how their surfactancy influence the ESI processes, challenge their handling in solution and choices of precursor-to-product ions for MSMS of e.g., structural PFAS isomers. The method has been used to discover PFASs in industrial blends and in extracts from food contact materials.     

Structural analysis of platycosides in Platycodi Radix by liquid chromatography/electrospray ionization-tandem mass spectrometry

Platycosides extracted from Platycodi Radix were analyzed by HPLC coupled with electrospray ionization multistage tandem mass spectrometry (HPLC/ESI-MS(n)). Predominant [M+Na](+) ions in positive mode and [M-H](-) ions in negative mode in the direct ESI-MS spectra of extract provided information on molecular weights, but minor components and isomers could not be discriminated. However, combining HPLC and ESI-MS(n), allowed eleven platycosides, including four acetylated platycodin isomers and two prosapogenines to be analyzed. During MS(2) analysis conducted to elucidate the structures of platycosides, fragment ions provided information on sugar moieties attached at C-28 of triterpene structure of the platycosides. Glycosidic bond cleavages at C-3 were revealed by fragment ions in MS(3) spectra. Some characteristic fragment ions not related to sugar bond cleavage revealed that an esterified triterpene is linked to sugars at C-28. The only sugar ring-cross cleavage corresponding to 90 Da in the negative MS(2) spectrum took place at an arabinosyl sugar moiety. By using HPLC/ESI-MS(n), three acetylated platycosides in Platycodi Radix extract were newly identified.     

Liquid chromatography/tandem mass spectrometry analysis of long-chain oxidation products of cardiolipin induced by the hydroxyl radical

The anionic phospholipid cardiolipin (CL) is found almost exclusively in the inner membrane of mitochondria, playing an important role in energy metabolism. Oxidation of CL has been associated with apoptotic events and various pathologies. In this study, electrospray ionization mass spectrometry coupled with liquid chromatography (LC/ESI-MS) was used to identify tetralinoleoyl-cardiolipin (TLCL) modifications induced by the OH(·) radical generated under Fenton reaction conditions (H(2)O(2) and Fe(2+)). The identified oxidation products of TLCL contained 2, 4, 6 and 8 additional oxygen atoms. These long-chain oxidation products were characterized by LC/ESI-MS/MS as doubly [M-2H](2-) and singly charged [M-H](-) ions. A detailed analysis of the fragmentation pathways of these precursor ions allowed the identification of hydroperoxy derivatives of CL. MS/MS analysis indicated that CL oxidation products with 4, 6 and 8 oxygen atoms have one fatty acyl chain bearing 4 oxygen atoms ([RCOO+4O](-)). Even when the TLCL molecule was oxidized by the addition of eight oxygen atoms, one of the acyl chains remained non-modified and one fatty acyl chain contained three or four oxygen atoms. This led us to conclude that under oxidative conditions by the OH(·) radical, the distribution of oxygens/peroxy groups in the CL molecule is not random, even when CL has the same fatty acyl chains in all the positions. Using mass spectrometry, the oxidation products have been unequivocally assigned, which may be useful for their detection in biological samples.     

Effect of protonation and deprotonation on the gas-phase reactivity of fluorinated 1,2,4-triazines

Positive and negative electrospray mass spectrometry (MS), in-time and in-space MS(n) experiments, high-resolution and accurate mass measurements obtained with an Orbitrap, together with density functional theory calculations have been used to study the gas-phase ion chemistry of a series of fluorinated 1,2,4-triazines. As a result of low-energy collision-induced dissociations, occurring in an ion trap and in a triple quadrupole, their protonated and deprotonated molecules show interesting features depending on the nature and structure of the precursor ions. The occurrence of elimination/hydration reactions produced by positive ions in the ion trap is noteworthy. Decompositions of deprotonated molecules, initiated by elimination of a hydroxyl radical from [M-H](-), are dominated by radical anions. Theoretical calculations have allowed us to obtain information on atom sites involved in the protonation and deprotonation reactions.     

Electron ionisation and electrospray ionisation mass spectrometric study of a series of isomeric methyl-, dimethyl- and trimethylalloxazines

Electron ionisation (EI) mass spectra and electrospray ionisation (ESI) mass spectra at different cone voltages of a series of isomeric methyl- and dimethylalloxazines are discussed, and compared with those of lumichrome, and 1- and 3-methyllumichrome. Examination of ESI mass spectra taken at a higher cone voltage and the use of isotope-labelled methanol allow us to discuss the fragmentation pathways of [M+H]+ and [M-H](-) ions. The fragmentation pathways of all of the compounds and the characteristic fragment ions formed in EI-MS are compared with published data. The influence of methyl and dimethyl substituents in the benzene ring on the fragmentation pathways leading to the loss of 43 and 45 Da upon both electron and electrospray ionisation is described.     

Structural characterization of iridoid glucosides by ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry

The mass spectral fragmentation behavior of ten iridoid glucosides (IGs) has been studied using electrospray ionization (ESI), collision-induced dissociation (CID), and quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS). In the negative ESI mass spectra, the deprotonated [M-H](-) ion was observed for all of the ten IGs except gardoside methyl ester, while the formate adduct [M+HCOO](-) ion appeared to be favored by the presence of a methyl ester or a lactone group in the C-4 position when formic acid was added to the mobile phase. The CID MS/MS spectra of the [M-H](-) ions have been used for structural elucidation. Ring cleavages of the aglycone moiety have been observed in the MS/MS spectra, corresponding to (1,4)F(-), (2,6)F(-), (2,7)F(-), and (2,7)F(0) (-) ions, based on accurate mass measurements and the elemental compositions of the product ions. These characteristic ions gave valuable information on the basic structural skeletons. Furthermore, on the basis of the relative abundances of the fragment ions (1,4)F(-) and (2,7)F(-), different sub-classes, such as cyclopentane-type and 7,8-cyclopentene-type IGs, can be differentiated. Ring cleavage of the sugar moieties was also observed, yielding useful information for their characterization. In addition, the neutral losses, such as H(2)O, CO(2), CH(3)OH, CH(3)COOH, and glucosidic units, have proved useful for confirming the presence of functional substituents in the structures of the IGs. Based on the fragmentation patterns of these standard IGs, twelve IGs have been characterized in an extract of Hedyotis diffusa Willd. by means of ultra-performance liquid chromatography/Q-TOF MS/MS, of which six have been unambiguously identified and the other six have been tentatively identified.     

Halogeno-substituted 2-aminobenzoic acid derivatives for negative ion fragmentation studies of N-linked carbohydrates

Negative ion electrospray mass spectra of high-mannose N-linked glycans derivatised with 2-aminobenzoic acids and ionised from solutions containing ammonium hydroxide gave prominent [M-H](-) ions accompanied by weaker [M-2H](2-) ions. Fragmentation of both types of ions gave prominent singly charged glycosidic cleavage ions containing the derivatised reducing terminus and ions from the non-reducing terminus that appeared to be products of cross-ring cleavages. Differentiation of these two groups of ions was conveniently achieved in a single spectrum by use of chloro- or bromo-substituted benzoic acids in order to label ions containing the derivative with an atom with a distinctive isotope pattern. Fragmentation of the doubly charged ions gave more abundant fragments, both singly and doubly charged, than did fragmentation of the singly charged ions, but information of chain branching was masked by the appearance of prominent ions produced by internal cleavages.     

Characterization of alpha- and gamma-glutamyl dipeptides by negative ion collision-induced dissociation

The low-energy CID mass spectra of the [M-H](-) ions of a variety of dipeptides containing glutamic acid have been obtained using cone-voltage collisional activation. Dipeptides with the gamma-linkage, H-Glu(Xxx-OH)-OH, are readily distinguished from those with the alpha-linkage, H-Glu-Xxx-OH, by the much more prominent elimination of H-Xxx-OH from the [M-H](-) ions of the former isomers, resulting in formation of m/z 128, presumably deprotonated pyroglutamic acid. Dipeptides with the reverse linkage, H-Xxx-Glu-OH, show distinctive fragmentation reactions of the [M-H](-) ions including enhanced elimination of CO(2) and formation of deprotonated glutamic acid. Exchange of the labile hydrogens for deuterium has shown that there is considerable interchange of C-bonded hydrogens with labile (N- and O-bonded) hydrogens prior to most fragmentation reactions. All dipeptides show loss of H(2)O from [M-H](-). MS(3) studies show that the [M-H-H(2)O](-) ion derived from H-Glu-Gly-OH has the structure of deprotonated pyroglutamylglycine while the [M-H-H(2)O](-) ions derived from H-Glu(Gly-OH)-OH and H-Gly-Glu-OH show a different fragmentation behaviour indicating distinct structures for the fragment ions.     

Characterization and quantification of soy isoflavone metabolites in serum of renal transplanted patients by high-performance liquid chromatography/electrospray ionization mass spectrometry

During a dietary intervention study on 16 renal transplanted patients, in which 25 g/day of animal proteins were replaced with 25 g of soy proteins, the metabolic profile of soy isoflavones in serum was characterized. This paper describes a reliable and fast liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) method, in negative ion mode, allowing the characterization and simultaneous quantification of several soy isoflavone metabolites. Six metabolites were identified and quantified: daidzein ([M-H](-) at m/z 252.8), dihydrodaidzein (DHD, [M-H](-) at m/z 254.8), equol ([M-H](-) at m/z 240.9), O-desmethylangolensin (O-DMA, [M-H](-) at m/z 256.8), genistein ([M-H](-) at m/z 268.8), and dihydrogenistein (DHG, ([M-H(+)](-) at m/z 270.8). Quantification was assessed using two deuterated internal standards, D(3)-daidzein and D(4)-genistein. This method permitted a limit of quantification (LOQ, S/N = 10) and a limit of detection (LOD, S/N = 3) of 0.05 microM and 0.005 microM for all analytes, except for genistein, where the LOQ and LOD were 0.005 microM and 0.001 microM, respectively. The linearity ranges were from 0.005 to 1.5 microM for genistein, from 0.05 to 1.5 microM for DHG, and from 0.05 to 0.7 microM for the other metabolites. The relative standard deviations (RSDs) were between 0.19% and 13.9% at the LOQ concentration for all metabolites, and between 0.6% and 4.8% at the maximum concentration. On the basis of the results obtained in the dietary intervention study, it was possible to split the patients into five groups characterized by different metabolic pathways.     

Characterization of carbofuran photodegradation by-products by liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry

The structural elucidation of by-products arising from carbofuran photodegradation using a high-pressure UV lamp has been investigated by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) employing a quadrupole time-of-flight mass spectrometer. Exact mass measurements of the [M + H]+ ions of the by-products and of product ions allowed the elemental formulae and related structures of seven photodegradation by-products (resulting, respectively, from photo-Fries rearrangement, hydroxylation of the benzene ring, oxidation of the 2,3-dihydrobenzofuran ring, cleavage of the carbamate group, hydrolysis of the ether group and the newly observed radical coupling and decarboxylation processes) to be determined confidently. Accurate mass measurements of product ions allowed ambiguities to be removed concerning neutral losses having the same nominal mass, namely CO and C2H4, allowing the fragmentation patterns to be rationalized.     

Lipid composition of membranes of Escherichia coli by liquid chromatography/tandem mass spectrometry using negative electrospray ionization

A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method using reversed-phase chromatography was developed for the analysis of phospholipids from bacterial extracts of a wild-type strain of Escherichia coli. Product ion mass spectra from [M--H](-) precursor ions allowed an identification of individual phospholipid species that includes both fatty acid composition and fatty acyl location on the glycerol backbone using diagnostic product ions. Thus, complete assignment, including sn-1/sn-2 fatty acyl position, was achieved for this strain of E. coli. In addition, the phospholipids were quantified relative to one another using an internal standard method.     

Improved liquid chromatography/mass spectrometric analysis of low molecular weight carboxylic acids by ion exclusion separation with electrospray ionization

An improved, simple and sensitive analytical method for low molecular weight organic acids has been developed. A mixture of acetic, propionic, butyric, glycolic, lactic, 2-hydroxybutyric, malonic, succinic, glutaric, tartaric and citric acids was separated on a semi-rigid styrene-divinylbenzene copolymer-based H-type cation-exchange resin (ULTRON PS-80H) based on an ion exclusion chromatographic (IEC) mechanism, with detection using electrospray ionization mass spectrometry (ESI-MS). Formic or acetic acid was used as a mobile phase to separate the carboxylic acids within 15 min. For liquid chromatography/mass spectrometry (LC/MS), the ESI interface was used in both positive and negative ionization mode. ESI produced reasonable signals from positive ions, [M+NH(4)](+), of acetic, propionic and butyric acids and from negative ions, [M-H](-), of glycolic, lactic, 2-hydroxybutyric, malonic, succinic, glutaric, tartaric and citric acids. The effects of ionization parameters, source temperature, capillary voltage and cone voltage, on sensitivity and linearity were examined. Linear plots of peak area versus concentration were obtained over the range 0.1-20 ppm for MS detection. The detection limits of the target carboxylic acids calculated at signal-to-noise (S/N) ratio of 3 ranged from 9 to 59 ppb. The reproducibility of retention times and peak areas were 0.55-1.25 and 0.85-2.45%, respectively.