Fragmentation of mycosporine-like amino acids by hydrogen/deuterium exchange and electrospray ionisation tandem mass spectrometry

The determination and identification of mycosporine-like amino acids (MAAs) from algae remain a major challenge due to the low concentration. Mass spectrometry (MS) can make an invaluable contribution in the search and identification of MAAs because of its high sensitivity, possibility of coupling with liquid chromatography, and the availability of powerful tandem mass spectrometric techniques. However, the unequivocal determination of the presence and location of important functional groups present on the basic skeleton of the MAAs is often elusive due to their inherent instability under MS conditions. In this study, the use of hydrogen/deuterium (H/D) exchange and electrospray ionisation tandem mass spectrometry (ESI-MS/MS) for characterisation of four MAAs (palythine, asterina, palythinol and shinorine) isolated from the macroalgae Gracilaria tenuistipitata Chang et Xia was investigated. The accurate-mass confirmation of the protonated molecules was performed on a Q-TOF instrument. We demonstrate that employing deuterium labelling in ESI-MS/MS analysis provides a convenient tool for the determination of new MAAs. Although the fragmentation patterns of MAAs were discussed earlier, to our knowledge, this is the first time that mechanisms are proposed.     

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.     

Rapid screening and identification of cytochalasins by electrospray tandem mass spectrometry

The cytochalasin class of fungal metabolites was analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) with the aim of developing a methodology for their rapid identification in microbial extracts. ESI-MS analyses of reference cytochalasins were performed and several product ions were produced in MS/MS experiments on parent ions that are structurally characteristic. A precursor ion search was performed to detect cytochalasins in an ethyl acetate extract of fungal strain RK97-F21. Three cytochalasins were detected and one of the components was identified as epoxycytochalasin H by comparing the tandem mass spectra of the product ions with those of reference compounds. This finding was further validated by LC/MS and LC/MS/MS experiments.     

Study of the mass spectrometric fragmentation of pseudouridine: comparison of fragmentation data obtained by matrix-assisted laser desorption/ionisation post-source decay, electrospray ion trap multistage mass spectrometry, and by a method utilising electrospray quadrupole time-of-flight tandem mass spectrometry and in-source fragmentation

Many nucleosides and their modified forms have been studied by mass spectrometry elaborating the detailed fragmentation pathways under MS2 and MS(n) conditions. Although the C-nucleoside pseudouridine has been fragmented and studied briefly, usually amongst many other nucleosides, it has not been investigated to the same extent as other nucleosides. In this report a number of different mass spectrometric techniques are applied to obtain a fuller picture of pseudouridine fragmentation. At the same time this study is used to compare different tandem mass spectrometric techniques, including a novel methodology utilising a quadrupole time-of-flight (Q-ToF) instrument for MS(n) analysis comparable with that available with an ion trap mass spectrometer.     

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.     

Collision-induced dissociation pathways of anabolic steroids by electrospray ionization tandem mass spectrometry

Anabolic steroids are structurally similar compounds, and their product-ion spectra obtained by tandem mass spectrometry under electrospray ionization conditions are quite difficult to interpret because of poly-ring structures and lack of a charge-retaining center in their chemical structures. In the present study, the fragmentation of nine anabolic steroids of interest to the racing industry was investigated by using triple quadrupole mass spectrometer, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, and a linear ion trap instrument. With the aid of an expert system software (Mass Frontier version 3.0), accurate mass measurements, and multiple stage tandem mass spectrometric (MS(n)) experiments, fragmentation pathways were elucidated for boldenone, methandrostenolone, tetrahydrogestrinone (THG), trenbolone, normethandrolone and mibolerone. Small differences in the chemical structures of the steroids, such as an additional double-bond or a methyl group, result in significantly different fragmentation pathways. The fragmentation pathways proposed in this paper allow interpretation of major product ions of other anabolic steroids reported by other researchers in a recent publication. The proposed fragmentation pathways are helpful for characterization of new steroids. The approach used in this study for elucidation of the fragmentation pathways is helpful in interpretation of complicated product-ion spectra of other compounds, drugs and their metabolites.     

Characterisation of oxazepam degradation products by high‐performance liquid chromatography/electrospray ionisation mass spectrometry and electrospray ionisation quadrupole time‐of‐flight tandem mass spectrometry

Oxazepam has been subjected to controlled degradation at 100°C for 3 h in 0.5 M HCl and 0.5 M NaOH. Following neutralisation of the degradation mixture and removal of salts by solid‐phase extraction (SPE), isocratic high‐performance liquid chromatography/mass spectrometry (HPLC/MS) using water/methanol (25:75 v/v) as the mobile phase was carried out using a flow diverter to collect fractions prior to their characterisation by electrospray ionisation multi‐stage mass spectrometry (ESI‐MSⁿ) and proposal of the corresponding fragmentation patterns. The elemental compositions of the degradation products and their MS fragments were evaluated using electrospray ionisation quadrupole time‐of‐flight tandem mass spectrometry (ESI‐QTOF‐MS/MS) which was then used to support the proposed fragmentation patterns. Copyright © 2010 John Wiley & Sons, Ltd.     

Identification of components in waste streams by electrospray and tandem mass spectrometry

Highly polar, non-gas-chromatographable compounds have few unambiguous analysis protocols for environmental applications. A recent environmental investigation, concerning the identification of a non-gas-chromatographable yellow component in chemical waste water and in effluents from a biological wastewater treatment plant required the use of a number of analytical approaches. Electrospray mass spectrometry, tandem mass spectrometry, high-performance liquid chromatography, nuclear magnetic resonance, and molecular spectroscopy of commercial and synthesized chlorodinitrophenol isomers were required in order to identify the specific isomer causing the color. The present report summarizes the electrospray ionization and tandem mass spectrometric studies that were used. The mass spectrometric study shows that two different isomers of chlorodinitrophenol exhibit very different collision-induced dissociation (CID) spectra. Differences in the tandem mass spectra can be attributed to the different structures of the anions formed from these two different isomers. Instrumentation that uses electrospray ionization and produces CID mass spectra and optical absorption spectra in a single analysis may be required in order to produce highly specific information on non-gas-chromatographable compounds found in the environment.     

Porphyrin profiles in blood, urine and faeces by HPLC/electrospray ionization tandem mass spectrometry

A high-resolution high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry method is described for the analysis of porphyrins in blood, urine and faeces. The gradient elution reversed-phase HPLC system using acetonitrile-methanol-1 m ammonium acetate/acetic acid buffer (pH 5.16) as gradient solvent mixtures was able to separate all porphyrin metabolites, including the type I and type III isomers of uroporphyrin, hepta-, hexa- and penta-carboxylic acid porphyrins and coproporphyrin. The porphyrins were positively identified by the protonated molecules [M+H](+) and further characterized by tandem mass spectrometric analysis with each porphyrin giving a characteristic collisioninduced dissociation product ion spectrum. The mass chromatograms obtained by HPLC/ESI MS are useful for the differential diagnosis of the porphyrias, since each type of porphyria has a typical porphyrin excretion pattern.     

Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds.     

Identification of the 'wrong' active pharmaceutical ingredient in a counterfeit Halfan drug product using accurate mass electrospray ionisation mass spectrometry,accurate mass tandem mass spectrometry and liquid chromatography/mass spectrometry

Methodology is presented for identifying an unknown active (pharmaceutical) ingredient (AI) in a counterfeit drug product. A range of mass spectrometric techniques, i.e., accurate mass mass spectrometry, tandem mass spectrometry (MS/MS) and liquid chromatography/mass spectrometry (LC/MS), has been employed to determine the AI in a counterfeit Halfan suspension, an antimalarial drug. In particular, use of LockSpray accurate mass MS/MS allowed identification of parts of the molecule from fragments, hence limiting the number of possible elemental compositions for the nominal mass of 278 found for the AI in the counterfeit product. The analysis of the isotope pattern observed for the protonated molecule further reduced the number of possible elemental compositions. A literature search for readily commercially available compounds of molecular formula C(12)H(14)N(4)O(2)S suggested that the AI was either sulfamethazine or sulfisomidine. An LC/MS separation of those two compounds and reference MS/MS spectra obtained for sulfamethazine and sulfisomidine led to the conclusion that the AI in the counterfeit Halfan suspension is sulfamethazine, which is an antibacterial agent.     

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.     

Investigation of the tris(trimethoxyphenyl)phosphonium acetyl charged derivatives of peptides by electrospray ionization mass spectrometry and tandem mass spectrometry

Charged derivatives of peptides are useful in obtaining simpler collision-activated dissociation (CAD) mass spectra. An N-terminal charge-derivatizing reagent capable of reacting with picomole levels of peptide has been recently reported (Huang et al. Anal. Chem. 1997, 69, 137-144) in the contexts of analyses by fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Electrospray ionization (ESI) mass spectrometric investigation of these tris(trimethoxyphenylphosphonium) acetyl derivatives are described in this article, including studies by in-source fragmentation (ISF) and tandem mass spectrometry (MS/MS). Results from ISF are compared with those from MS/MS. Similarities and differences between ESI-ISF, MALDI-post-source decay (PSD), and FAB-CAD data are presented. Differences in fragmentation of these charged derivatives in the triple quadrupole and ion trap mass spectrometers also are discussed. Application of this derivatizing procedure to tryptic digests and subsequent analysis by liquid chromatography-mass spectrometry is also shown.     

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.     

Identification of new minor metabolites of penicillin G in human serum by multiple-stage tandem mass spectrometry

Liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) were applied to characterize drug metabolites. Although these two methods have overcome the identification and structural characterization of metabolites analysis, they remain time‐consuming processes. In this study, a novel multiple‐stage tandem mass spectrometric method (MSⁿ) was evaluated for identification and characterization of new minor metabolism profiling of penicillin G, one of the β‐lactam antibiotics, in human serum. Seven minor metabolites including five phase I metabolites and two phase II metabolites of penicillin G were identified by using data‐dependent LC/MSⁿ screening in one chromatographic run. The accuracy masses of seven identified metabolites of penicillin G were also confirmed by mass spectral calibration software (MassWorks?). The proposed data‐dependent LC/MSⁿ method is a powerful tool to provide large amounts of the necessary structural information to characterize minor metabolite in metabolism profiling. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of native and chemically modified oligonucleotides by tandem ion-pair reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry

Ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) was utilized in tandem with negative-ion electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) for the analysis of native and chemically modified oligonucleotides. Separation was performed on a 1.0 x 50 mm column packed with porous C(18) sorbent with a particle size of 2.5 microm and an average pore diameter of 140 A. A method was developed which maximizes both chromatographic separation and mass spectrometric sensitivity using an optimized buffer system containing triethylamine and 1,1,1,3,3,3-hexafluoro-2-propanol with a methanol gradient. The ESI-TOFMS tuning parameters were also optimized in order to minimize in-source fragmentation and achieve the best sensitivity. Analyses of native, phosphorothioate, and guanine-rich oligonucleotides were performed by LC/MS. Detection limits were at sub-picomole levels with an average mass accuracy of 125 ppm. The described method allowed for the LC/MS analysis of oligonucleotides up to 110mer in length with little alkali cation adduction. Since sensitive detection of oligonucleotides was achieved with ultraviolet (UV) detection, we utilized a combination of UV-MS for quantitation (UV) and characterization (MS) of oligonucleotides and their failure sequence fragments/metabolites.     

Peptide mapping with mobile phases of intermediate pH value using capillary reversed-phase high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry

This investigation describes the separation of tryptic peptides by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) with eluents in the intermediate pH range, followed by in-line electrospray ionisation tandem mass spectrometry (ESI-MS/MS) analysis. For these purposes, gradient elution procedures with an aqueous eluent containing 20 mM ammonium formate, and an increasing content of acetonitrile or methanol, were employed. Compared to the analysis of the same tryptic peptides under low-pH conditions with an ion-pairing reagent, the increase in the pH with the 20 mM ammonium formate mobile phase led to significant changes in both peptide retention to the reversed-phase column and the collision-induced dissociation at the MS/MS stage as a consequence of the changes in the physico-chemical properties of these peptides, such as their overall charge, polarity and relative hydrophobicity. Thus, improved selectivity for the peptide separation and favourable tandem mass spectrometry analysis could be obtained with eluents in this intermediate pH range. The number of tryptic peptides identified by the new approach for the proteins investigated were significantly higher than that obtained by the conventional low-pH methods. Moreover, analysis of protein digests at very low concentrations was also performed under both acidic and intermediate pH conditions and similar improvements in selectivity and MS/MS detection limits were observed, i.e. identification of more distinct peptides and higher sequence coverage of the protein was obtained when eluents of intermediate pH were employed. This study therefore highlights the potential of conducting peptide mapping in the intermediate pH range to achieve more reliable and sensitive protein identifications with capillary RP-HPLC–ESI-MS/MS.     

Determination of linkage position and anomeric configuration in Hex-Fuc disaccharides using electrospray ionization tandem mass spectrometry

Fixed-energy sequential tandem mass spectrometry (MS(n)) capabilities offered by quadrupole ion trap instruments have been explored in a systematic study of six isomers of Gal-Fucalpha-OBenzyl disaccharides. Under collision-induced dissociation (CID), sodiated molecular species generated in the positive-ion electrospray ionization mode yield simple and predictable mass spectra. Information on interglycosidic linkages and configurations can be deduced from the relative intensities of the selected diagnostic fragments arising from the glycosidic bond cleavages and corroborated by the fragments arising from cross-ring cleavages. As the CID patterns are not dependent on the number of prior tandem mass spectrometric steps, structures can be unambiguously assigned by matching the spectra with a library. The rules governing the fragmentation behavior of this class of oligosaccharides were tested for a representative isomeric disaccharide, Glcbeta1,3Fucalpha-OAllyl. The findings establish a basis for using MS(n) with a quadrupole ion trap instrument to elucidate structures of hexose-fucose subunits from more complicated oligosaccharides. Energy-resolved mass spectra were also acquired by CID tandem triple-quadrupole mass spectrometry. The breakdown behavior of the molecular ions revealed patterns which could differentiate stereoisomers of Gal-Fuc disaccharides over a range of collision energy from 20 to 50 eV.     

Comparison of flow injection analysis electrospray mass spectrometry and tandem mass spectrometry and electrospray high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry for the determination of underivatized amino acids

Twenty proteinogenic amino acids (AAs) were determined without derivatization using flow injection analysis followed by electrospray ionization mass spectrometry and tandem mass spectrometry (ESI-MS and ESI-MS/MS) and electrospray ionization high-field asymmetric waveform ion mobility mass spectrometry and tandem mass spectrometry (ESI-FAIMS-MS and ESI-FAIMS-MS/MS), in positive and negative ionization modes. Three separate sets of ESI-FAIMS conditions were used for the separation and detection of the 20 AAs. Typically ESI-FAIMS-MS showed somewhat improved sensitivity and significantly better signal-to-noise ratios than ESI-MS mainly due to the elimination of background noise. However, the difference between ESI-FAIMS-MS and ESI-MS/MS was significantly less. ESI-FAIMS was able to partially or completely resolve all the isobaric amino acid overlaps such as leucine, isoleucine and hydroxyproline or lysine and glutamine. Detection limits for the amino acids in ESI-FAIMS-MS mode ranged from 2 ng/mL for proline to 200 ng/mL for aspartic acid. Overall, ESI-FAIMS-MS is the preferred method for the quantitative analysis of AAs in a hydrolyzed yeast matrix.     

Gas phase reactivity of isomeric arylglycosides towards amines. A chemical ionization mass spectrometry and tandem mass spectrometry study

Chemical ionization mass spectrometry (MS) and tandem mass spectrometry (MS/MS) experiments have been performed for the structural characterization and isomeric differentiation of two series of C- and O-linked arylglycosides with potential antioxidant activity. Different amines have been used for producing gas phase chemical ionization. Depending on their proton affinity and steric hindrance, adduct ions with different stability are formed. The most stable adducts are produced by ethylamine and they have been extensively structurally characterized by experimental and theoretical approaches. Energy resolved chemical ionization tandem mass spectrometric experiments have allowed unambiguous characterization and differentiation of both the anomers differing at the configuration of the glycosidic C(1) atom, and regio- and structural isomers at extremely low concentrations, typical of mass spectrometry. This study has shown that amine chemical ionization mass spectrometry and MS/MS are powerful and versatile tools for the structural characterization of arylglycosides.