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.     

Rapid accurate mass desorption electrospray ionisation tandem mass spectrometry of pharmaceutical samples

Desorption electrospray ionisation (DESI) has been successfully combined with a hybrid quadrupole time-of-flight mass spectrometer to provide mass spectra and product ion mass spectra of active ingredients formulated in pharmaceutical tablets, gels and ointments. Accurate mass data has been obtained from the DESI mass spectra and of the product ion fragments of selected ions, greatly enhancing the selectivity and information content of the experiment. This accurate mass information only takes seconds to acquire since the DESI technique does not require any sample preparation or extraction prior to mass analysis.     

The rapid characterisation of poly(ethylene glycol) oligomers using desorption electrospray ionisation tandem mass spectrometry combined with novel product ion peak assignment software

A rapid method for the characterisation of polyglycol esters and ethers is described which uses accurate mass desorption electrospray ionisation (DESI) quadrupole time-of-flight mass spectrometry (Q-ToFMS). The results are combined with newly developed software which aids the interpretation of product ions produced using collision-induced dissociation (CID) of selected precursor ions. The poly(ethylene glycol) (PEG) samples analysed were PEG dibenzoate, PEG monooleate, PEG butyl ether, PEG bis(2-ethyl hexanoate) and PEG diacrylate. Lithium metal was used for cationisation of the PEG oligomers since it yielded the most useful structural information compared with other group I metals. The full scan mass spectra and product ion mass spectra were all obtained in <5 s. Interpretation of the MS/MS product ion spectra, using the product ion interpretation software which incorporates previously developed fragmentation rules, was carried out in <1 s.     

Structural characterisation of underivatised olive pulp xylo-oligosaccharides by mass spectrometry using matrix-assisted laser desorption/ionisation and electrospray ionisation

Purified olive pulp glucuronoxylans, with a Xyl/GlcA ratio of 7:1, were subjected to mild acid hydrolysis and the mixture of oligosaccharides obtained was fractionated by size exclusion chromatography. One elution fraction representative of low molecular weight oligosaccharides was analysed by mass spectrometry using matrix-assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI) as ionisation methods, in the positive mode. Both types of spectra showed cationised molecules [M + Na](+) of xylo-oligosaccharides in a range below m/z 1,000. The xylo-oligosaccharide structures identified were series of neutral oligosaccharides of xylose (Xyl(n), n = 3-7), of acidic oligosaccharides substituted by one glucuronic acid (Xyl(n)GlcA, n = 3-5) and by two glucuronic acid residues (Xyl(n)GlcA(2), n = 2 and 3), and also of acidic oligosaccharides substituted with one 4-O-methylglucuronic acid residue (Xyl(n)meGlcA, n = 2-4). The proposed structures were confirmed by tandem mass (MS/MS) spectra obtained using collision induced dissociation of the molecular ions. Fragmentation of cationised adducts of neutral Xyl(n) yielded C- and A-type fragments, while ammonium adducts mainly yielded B-type fragments. The fragmentation of the sodium adducts of acidic oligosaccharides (Xyl(n)meGlcA, Xyl(n)GlcA) resulted in the loss of the substituting residue (GlcA or meGlcA) as the predominant fragment, while the corresponding ammonium adducts yielded B-type fragments.     

Analysis of tryptic peptides using desorption electrospray ionisation combined with ion mobility spectrometry/mass spectrometry

A novel method is reported for rapid protein identification by the analysis of tryptic peptides using desorption electrospray ionisation (DESI) coupled with hyphenated ion mobility spectrometry and quadrupole time-of-flight mass spectrometry (IMS/Q-ToF-MS). Confident protein identification is demonstrated for the analysis of tryptically digested bovine serum albumin (BSA), with no sample pre-treatment or clean-up. Electrophoretic ion mobility separation of ions generated by DESI allowed examination of charge-state and mobility distributions for tryptic peptide mixtures. Selective interrogation of singly charged ions allowed isobaric peptide responses to be distinguished, along with a reduction in spectral noise. The mobility-selected singly charged peptide responses were presented as a pseudo-peptide mass fingerprint (p-PMF) for protein database searching. Comparative data are shown for electrospray ionisation (ESI) of the BSA digest, without sample clean-up, from which confident protein identification could not be made. Implications for the robustness of the DESI method, together with potential insights into mechanisms for DESI of proteolytic digests, are discussed.     

Capillary liquid chromatography/atmospheric-pressure matrix-assisted laser desorption/ionisation ion trap mass spectrometry: a comparison with liquid chromatography/matrix-assisted laser desorption/ionisation time-of-flight and liquid chromatography/electrospray ionisation quadrupole time-of-flight for the identification of tryptic peptides

The atmospheric-pressure matrix-assisted laser desorption/ionisation quadrupole ion trap (AP-MALDI-QIT) analysis of tryptic peptides is reported following capillary liquid chromatographic (LC) separation and direct analysis of a protein digest. Peptide fragments were identified by peptide mass fingerprinting from mass spectrometric data and sequence analysis obtained by tandem mass spectrometry of the principal mass spectral peaks using a data-dependent scanning protocol. These data were compared with those from mass spectrometric analysis using capillary LC/MALDI-time-of-flight (TOF) and capillary LC/electrospray ionisation (ESI)-quadrupole TOF. For all three configurations the resulting data were searched against the MSDB database, using MASCOT and the sequence coverage compared for each technique. Complementary data were obtained using the three techniques.     

Top-down protein sequencing by CID and ECD using desorption electrospray ionisation (DESI) and high-field FTICR mass spectrometry

We report high resolution spectra for the medium molecular weight proteins myoglobin and cytochrome-c obtained using a custom desorption electrospray ionisation (DESI) source coupled to a Bruker Daltonics 12 T Apex Qe Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). The DESI source was designed for accurate alignment and reproduction of critical geometric variables. A two axis motorised stage was included to enable automated rastering of the sample under the DESI plume. Spectra for the intact proteins have been obtained under single-acquisition conditions and a top-down analysis of cytochrome-c was performed using both collision induced dissociation (CID) and electron capture dissociation (ECD) of the isolated [M+15H]¹⁵⁺ charge state. The sequence coverage is comparable to that obtained using electrospray ionisation, demonstrating the utility of top-down protein analysis by DESI FTICR-MS.     

Collision-induced fragmentation pathways including odd-electron ion formation from desorption electrospray ionisation generated protonated and deprotonated drugs derived from tandem accurate mass spectrometry

The rapid desorption electrospray ionisation (DESI) of some small molecules and their fragmentation using a triple-quadrupole and a hybrid quadrupole time-of-flight mass spectrometer (Q-ToF) have been investigated. Various scanning modes have been employed using the triple-quadrupole instrument to elucidate fragmentation pathways for the product ions observed in the collision-induced dissociation (CID) spectra. Together with accurate mass tandem mass spectrometry (MS/MS) measurements performed on the hybrid Q-ToF mass spectrometer, unequivocal product ion identification and fragmentation pathways were determined for deprotonated metoclopramide and protonated aspirin, caffeine and nicotine. Ion structures and fragmentation pathway mechanisms have been proposed and compared with previously published data. The necessity for elevated resolution for the differentiation of isobaric ions are discussed.     

Desorption electrospray ionisation mass spectrometry and tandem mass spectrometry of low molecular weight synthetic polymers

A range of low molecular weight synthetic polymers has been characterised by means of desorption electrospray ionisation (DESI) combined with both mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Accurate mass experiments were used to aid the structural determination of some of the oligomeric materials. The polymers analysed were poly(ethylene glycol) (PEG), polypropylene glycol (PPG), poly(methyl methacrylate) (PMMA) and poly(alpha-methyl styrene). An application of the technique for characterisation of a polymer used as part of an active ingredient in a pharmaceutical tablet is described. The mass spectra and tandem mass spectra of all of the polymers were obtained in seconds, indicating the sensitivity of the technique.     

Energy-dependent electrospray ionisation mass spectrometry: applications in transition metal carbonyl chemistry

Maps of electrospray mass spectrometry data, plotted as cone voltage versus mass-to-charge ratio, provide a clear and compact method of visualising the accompanying fragmentation processes, in this case applied to the sequential removal of ligands from transition-metal carbonyl complexes. The technique is described as energy-dependent electrospray ionisation mass spectrometry (EDESI-MS). Copyright 2000 John Wiley & Sons, Ltd.     

Ionic liquids enable electrospray ionisation mass spectrometry in hexane

Addition of a lipophilic ionic liquid to non-polar, hydrocarbon solvents (such as hexane and toluene) permits electrospray ionisation mass spectrometric analysis of dissolved analytes.     

Letter: characterisation and identification of spermine and spermidine derivatives in Microdesmis keayana and Microdesmis puberula roots by electrospray ionisation tandem mass spectrometry and high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry

Three new N(1),N(5),N(14)-tris(4- hydroxycinnamoyl)spermines were identified in hydromethanolic root extracts of Microdesmis keayana J. Léonard and Microdesmis puberula Hook f. The electrospray ionisation tandem mass spectrometry (ESI-MS/MS) technique with specific nuclear magnetic resonance analysis of hydrolysed products made it possible to identify N(1),N(5),N(14)-tris(p-coumaroyl)spermine, N(1)-feruloyl,N(5),N(14)-di(p-coumaroyl)spermine and N(1),N(5),N(14)-tris(feruloyl)spermine, named keayanines B, C and D, respectively. ESI-MS/MS analysis most effectively provided structural data although high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry was also used to characterise four other compounds from Microdesmis puberula-keayanidines A, B, C and keayanine A-which had already been identified in M. keayana. This chemical data is the first to be published for M. puberula which is a commonly used plant in Central African traditional medicine.     

On-line size-exclusion chromatography/electrospray ionisation mass spectrometry of aquatic humic and fulvic acids

Isolated aquatic humic and fulvic acids were analysed with on-line size exclusion chromatography/electrospray ionisation mass spectrometry (SEC/ESI-MS). An eluent composition which enabled electrospray ionisation was identified. The SEC separation improved interpretability of mass spectra and may open up new possibilities for molecular weight determination of humic substances. A linear dose-response relationship over a factor of 20 was obtained and the limit of detection was 50ng/uL for humic and fulvic acids. Spectral changes due to different ionisation conditions (pH and cone voltage) were investigated. A natural water sample from a Swedish lake was analysed. Copyright 2000 John Wiley & Sons, Ltd.     

Investigation of the ionisation and fragmentation behaviour of different nitroaromatic compounds occurring as polar metabolites of explosives using electrospray ionisation tandem mass spectrometry

In order to develop a liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) method for identification and quantification of polar metabolites of explosives using a triple quadrupole system, the mass spectrometric ionisation and fragmentation behaviour of different nitrophenols, nitro- and aminonitrobenzoic acids, nitrotoluenesulfonic acids, and aminonitrotoluenes was investigated. Due to their different molecular structures, the substances concerned showed a very different ionisation efficiency in the ESI process. Interestingly, 2,4-dinitrobenzoic acid yielded no mass signals in the Q1 scan suggesting a thermal decarboxylation in the ion source, whereas the corresponding 3,5-isomer showed a high ionisation yield. Using negative ionisation polarity, carboxylic, phenolic, and sulfonic acid groups were deprotonated resulting in molecular anions, which could be fragmented in a collision cell. A pronounced dependency of the produced fragment ion series on the kind and position of substituents at the nitrobenzene ring (ortho effects) was observed and exploited for the development of substance-specific detection methods in the multiple reaction monitoring mode. In case of benzoic and sulfonic acids, decarboxylation and desulfonation, respectively, were observed as the most frequent fragmentation reactions. Furthermore, besides loss of NO(2), NO fragmentation occurred and preceded a decarbonylation of the benzene ring. The expulsion of the open-shell molecules NO and NO(2) led to a variety of distonic radical anions.     

Differential ionisation of natural antioxidant polyenes in electrospray and nanospray mass spectrometry

Carotenoids are natural products with high economic relevance for the pharmaceutical industries and are a common subject for biochemical research. Reported here is a comparative study of the ionisation of carotenoids by electrospray mass spectrometry (ESI-MS) and nanospray mass spectrometry (nanoESI-MS). The results demonstrate that, along with solvent choice, the influence of the different ionisation processes of ESI and nanoESI are fundamental in determining how ionisation is achieved and which ions (molecular ion or protonated molecule) are observed in MS. The increased understanding afforded by this study will help in the development of unequivocal microanalytical methods for carotenoids and related antioxidant polyenes.     

Characterisation of humic substances using atmospheric pressure chemical ionisation and electrospray ionisation mass spectrometry combined with size-exclusion chromatography

Humic substances were analysed by atmospheric pressure chemical ionisation (APCI) and electrospray ionisation (ESI) mass spectrometry in positive and negative modes. Using APCI the average m/z range of humic substances was reduced 5-fold compared to ESI. High-resolution time-of-flight mass spectrometry revealed the formation of multiply charged molecules in the ESI mode. Moreover, it was possible to obtain daughter ion mass spectra of humic substances by nanospray tandem mass spectrometry. The size-exclusion chromatography elution profile of humic substances was highly influenced by the pH of the analyte solution. By contrast, the pH had no significant influence on the observed mass spectra of humic substances.     

A novel approach for identification and measurement of hemoglobin adducts with 1,2,3,4-diepoxybutane by liquid chromatography/electrospray ionisation mass spectrometry and matrix-assisted laser desorption/ionisation tandem mass spectrometry

The structural characterisation of the adducts formed by in vitro interaction of hemoglobin (Hb) with 1,2,3,4-diepoxybutane (DEB), the most reactive 1,3-butadiene (BD) metabolite, was obtained by liquid chromatography/electrospray ionisation mass spectrometry (LC/ES-MS) analysis of modified tryptic peptides of human hemoglobin chains. The reactive sites of human hemoglobin towards DEB and its hydroxylated derivatives (trihydroxybutyl (THB)-derivatives) were identified through the characterisation of alkylated tryptic peptides by matrix-assisted laser desorption/ionisation tandem mass spectrometry (MALDI-MS/MS). Based on this characterisation, a procedure was set up to measure the Hb-adducts of THB-derivatives by isotope dilution mass spectrometry with the use of a deuterated peptide standard. The results obtained here could permit optimisation of molecular dosimetry of BD-adducts, and extension of the analysis to the biological monitoring of occupational exposure to butadiene.     

Characterisation of botulinum toxins type A and B,by matrix-assisted laser desorption ionisation and electrospray mass spectrometry

A method earlier developed for the mass spectrometric (MS) identification of tetanus toxin (TTx) was applied to botulinum toxins type A and B (BTxA and BTxB). Botulinum toxins are extremely neurotoxic bacterial toxins, likely to be used as biological warfare agent. Biologically active BTxA and BTxB are comprised of a protein complex of the respective neurotoxins with specific haemagglutinins (HAs) and non-toxic non-haemagglutinins (NTNHs). These protein complexes are also observed in mass spectrometric identification. The particular BTxA complex, from Clostridium botulinum strain 62A, almost completely matched database data derived from genetic sequences known for this strain. Although no such database information was available for BTxB, from C. botulinum strain okra, all protein sequences from the complex except that of HA-70 were found to match proteins known from other type B strains. It was found that matrix-assisted laser desorption ionisation MS provides provisional identification from trypsin digest peptide maps and that liquid chromatography electrospray (tandem) mass spectrometry affords unequivocal identification from amino acid sequence information of digest peptides obtained in trypsin or pepsin digestion.     

The characterisation of selected antidepressant drugs using electrospray ionisation with ion trap mass spectrometry and with quadrupole time-of-flight mass spectrometry and their determination by high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry

The electrospray ionisation ion trap tandem mass spectrometry (ESI-MS(n)) of selected antidepressant drugs, i.e., citalopram, fluoxetine, mirtazapine, paroxetine, sertraline, and venlafaxine, has been investigated. Sequential product ion fragmentation experiments (MS(n)) have been performed in order to elucidate the degradation pathways for the [M+H](+) ions and their predominant product ions. These MS(n) experiments show certain characteristic fragmentations in that functional groups are generally cleaved from the ring systems as molecules such as H(2)O, amines and phenols. When an aromatic entity is present in a drug molecule together with a nitrogen-containing saturated ring structure as with mirtazapine, fragmentation initially occurs at the latter ring with the former being predictably resistant to fragmentation. Also, when an amine-containing drug molecule such as fluoxetine also contains a functional group, which liberates a phenol with a significantly lower DeltaH(f) (0) value than that of the corresponding amine, the phenol is preferentially liberated. The structures of product ions proposed for ESI-MS(n) can be supported by electrospray ionisation quadrupole-time-of-flight tandem mass spectrometry (ESI-QToF-MS/MS). These molecules can be identified and determined in mixtures at low ng/mL concentrations by the application of high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry (HPLC/ESI-MS(2)), which can also be used for their analysis in hair samples.     

Reversed-phase ion-pair chromatography coupled to electrospray ionisation mass spectrometry by on-line removal of the counter-ions.

A strong anion-exchanger was used as a trapping column to perform on-line coupling of ion-pair chromatography with electrospray ionisation mass spectrometry. Ion-pairing reagents were used to retain polar analytes of low molecular mass away from the solvent front in reversed-phase LC. The trapping column enabled removal of the non-volatile counter-ions from the mobile phase prior to detection, so that the electrospray process could be performed with favourable ionisation conditions and without contamination of the interface. The efficiency of the trapping process was studied for 1-octanesulfonic acid and sodium dodecyl sulfate as ion-pairing reagents. Using this on-line trapping method, biopterin and guanidine could be retained with a k'>2 and detected by electrospray mass spectrometry with a stable signal.