Structural characterization of hexoses and pentoses using lead cationization. An electrospray ionization and tandem mass spectrometric study

The analytical potential of the complexation of isomeric underivatized hexoses (D-glucose, D-galactose, D-mannose, D-talose, D-fructose), methylglycosides (1-O-methyl-alpha-D-glucose and 1-O-methyl-beta-D-glucose) and pentoses (D-ribose, D-xylose, D-arabinose and D-lyxose) by Pb(2+) ions, was investigated by electrospray ionization and tandem mass spectrometry (MS/MS). Pb(2+) ions react mainly with monosaccharides by proton abstraction to generate [Pb(monosaccharide)(m) - H](+) ions (m = 1-3). At low cone voltage, a less abundant series of doubly charged ions of general formula [Pb(monosaccharide)(n)](2+) is also observed. The maximum number n of monosaccharides surrounding a single Pb(2+) ion depends on the metal : monosaccharide ratio. Our study shows that MS/MS experiments have to be performed to differentiate Pb(2+)-coordinated monosaccharides. Upon collision, [Pb(monosaccharide) - H](+) species mainly dissociate according to cross-ring cleavages, leading to the elimination of C(n)H(2n)O(n) neutrals. The various fragmentation processes observed allow the C(1), C(2) and C(4) stereocenters of aldohexoses to be characterized, and also a clear distinction aldoses and fructose. Furthermore, careful analysis of tandem mass spectra also leads to successful aldopentose distinction. Lead cationization combined with MS/MS therefore appears particularly useful to identify underivatized monosaccharides.     

Characterization of dinitroporphyrin zinc complexes by electrospray ionization tandem mass spectrometry. Unusual fragmentations of beta-(1,3-dinitroalkyl) porphyrins

The zinc complexes of diaryl bis(p-nitrophenyl)porphyrins and beta-(1,3-dinitroalkyl)tetraphenylporphyrins were studied by electrospray ionization (ESI) tandem mass spectrometry (MS/MS). All porphyrins showed the protonated molecule under ESI conditions. The protonated molecules were induced to fragment and the corresponding ESI tandem mass spectra were analysed. Porphyrins with two p-nitrophenyl groups showed, as expected, characteristic fragmentations including either loss of one nitro group, as the major fragment of the tandem mass spectra, and loss of both nitro groups. In contrast, MS/MS of the beta-(1,3-dinitroalkyl)porphyrins provided interesting and unexpected results such as the absence (or in insignificant abundance) of the ions formed by loss of one nitro group. However, these porphyrins show an abundant fragment due to combined loss of the two nitro groups. Also, the typical beta-cleavage of the alkyl chain is not observed per se, only when combined with loss of HNO2 or *NO2. Instead, alpha-cleavage, with loss of the beta-pyrrolic substituent, is the most favourable process.     

Characterization of non-covalent complexes of rutin with cyclodextrins by electrospray ionization tandem mass spectrometry

Electrospray ionization tandem mass spectrometry (ESI-MS(n)) and the phase solubility method were used to characterize the gas-phase and solution-phase non-covalent complexes between rutin (R) and alpha-, beta- and gamma-cyclodextrins (CDs). The direct correlation between mass spectrometric results and solution-phase behavior is thus revealed. The order of the 1 : 1 association constants (K(c)) of the complexes between R and the three CDs in solution calculated from solubility diagrams is in good agreement with the order of their relative peak intensities and relative collision-induced dissociation (CID) energies of the complexes under the same ESI-MS(n) condition in both the positive and negative ion modes. Not only the binding stoichiometry but also the relative stabilities and even binding sites of the CD-R complexes can be elucidated by ESI-MS(n). The diagnostic fragmentation of CD-R complexes, with a significant contribution of covalent fragmentation of rutin leaving the quercetin (Q) moiety attached to the CDs, provides convincing evidence for the formation of inclusion complexes between R and CDs. The diagnostic fragment ions can be partly confirmed by the complexes between Q and CDs. The gas-phase stability order of the deprotonated CD-R complexes is beta-CD-R > alpha-CD-R > gamma-CD/R; beta-CD seems to bind R more strongly than the other CDs.     

Effect of metal cationization on the tandem mass spectra of glycosyl dithioacetals

The effect of metal cationization on the tandem mass spectra of glycosyl dithioacetals of glucose, mannose, galactose, rhamnose, arabinose and xylose was studied by electrospray ionization mass spectrometry under ammonium and metal (Li, Na, Ag and Cu) ion cationization conditions. The ammonium-cationized glycosyl dithioacetals fragment by loss of ammonia followed by either two molecules of EtSH or one molecule of EtSH and one molecule of H2O. Lithium cationization leads to additional eliminations such as EtSEt and EtSSEt and C-C cleavages. Elimination of EtSH is not observed under sodium cationization. Silver cationization, on the other hand, leads to additional fragmentations involving the elimination of silver as AgOH and AgSEt. Copper cationization results in adducts where copper has undergone a change of oxidation state from II to I. Li+, Ag+ and Cu+ cationization seem to favour cyclization resulting in elimination of EtSH. However, the mechanisms seem to be differently affected by different metal ions. Li+ and Ag+ cationization appear to be non-specific and favour cyclization involving C2-, C4- and C5-hydroxyl hydrogens, whereas Cu+ cationization seems to favour cyclization involving C4-hydroxyl hydrogen.     

Studies on the non-covalent complexes between oleanolic acid and cyclodextrins using electrospray ionization tandem mass spectrometry

Non-covalent inclusion complexes formed between an anti-inflammatory drug, oleanolic acid (OA), and alpha-, beta- and gamma-cyclodextrins (CDs) were investigated by means of solubility studies and electrospray ionization tandem mass spectrometry (ESI-MS(n)). The order of calculated association constants (K(1 : 1)) of complexes between OA and different CDs in solution is in good agreement with the order of their relative peak intensities and the relative CID energies of the complexes under the same ESI-MS(n) conditions. These results indicate a direct correlation between the behaviors of solution- and gas-phase complexes. ESI-MS can thus be used to evaluate solution-phase non-covalent complexes successfully. The experimental results show that the most stable 1 : 1 inclusion complexes between three CDs and OA can be formed, but 2 : 1 CD-OA complexes can be formed with beta- and gamma-CDs. Multi-component complexes of alpha-CD-OA-beta-CD (1 : 1 : 1), alpha-CD-OA-gamma-CD (1 : 1 : 1) and beta-CD-OA-gamma-CD (1 : 1 : 1) were found in equimolar CD mixtures with excess OA. The formation of 2 : 1 and multi-component 1 : 1 : 1 non-covalent CD-OA complexes indicates that beta- and gamma-CD are able to form sandwich-type inclusion non-covalent complexes with OA. The above results can be partly supported by the relative sizes of OA and CD cavities by molecular modeling calculations. All the complexes allow the detection of gaseous deprotonated CD-OA complexes in the negative ion mode at high abundances. The relative stabilities of the CDs-OA inclusion complexes in the gas phase can be evaluated from the relative CID energies in the ion trap (alpha-CD-OA < beta-CD-OA < gamma-CD-OA) in the negative ion mode.     

Determination of binding sites in carboplatin-bound cytochrome c using electrospray ionization mass spectrometry and tandem mass spectrometry

Interaction of carboplatin with cytochrome c (Cyt. c) has been investigated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). ESI-MS studies revealed that the ring-opened adducts of carboplatin with Cyt. c were formed in the stoichiometric ratio of 1:1 and 2:1 at pH 5.0 and 37 degrees C and in the stoichiometric ratio of 1:1 only at pH 7.0 and 37 degrees C. It was also found that Cyt. c could be cleaved by carboplatin at pH 2.5 and 50 degrees C. The cleaved fragments of Cyt. c were determined by ESI-MS and MS/MS analysis to be Glu66 approximately Met80, Ac-Gly01 approximately Met65, Glu66 approximately Glu104, Ac-Gly01 approximately Met80 and Ile81 approximately Glu104. The carboplatin prefers to anchor to Met65 first, then to Met80. To further confirm the binding site of Met, AcMet-Gly was used as the model molecule to investigate its interaction with carboplatin and its hydrolysis reaction. On the basis of species detected during the reaction monitored by ESI-MS, a possible pathway of the cleavage reaction was proposed.     

Characterization of aconitine-type alkaloids in the flowers of Aconitum kusnezoffii by electrospray ionization tandem mass spectrometry

The fragmentation mechanism of aconitine-type alkaloids in the flowers of Aconitum kusnezoffii (FAK) was investigated using electrospray ionization tandem mass spectrometry (ESI-MS(n)) firstly. The analysis of the collision-induced dissociation (CID) spectra of three purified aconitine standards and six previously reported aconitines indicated that the fragmentation of the protonated aconitines at low-energy CID follows a similar pathway. The elimination of a C(8)-substituent such as an acetic acid or a fatty acid is the dominant fragmentation mode in MS2. Successive losses of CH(3)COOH, CH(3)OH, H(2)O, BzOH, and CO are the main fragmentation pathways of aconitine-type alkaloids in MS(3) spectra. Based on these features, a rapid method for the direct detection and characterization of alkaloids from an ethanolic extract of FAK is described. All the known aconitum alkaloids are detected and a series of lipo-aconitines has been found for the first time in this plant.     

Structural elucidation and identification of alkaloids in Rhizoma Coptidis by electrospray ionization tandem mass spectrometry

Simple, convenient, sensitive and accurate analytical methods are needed for the structural characterization and identification of alkaloid components in Rhizoma Coptidis in traditional Chinese herbal medicine, which has important bioactivity. In this work, the identification of alkaloid compounds in Rhizoma Coptidis was investigated by obtaining molecular mass information using electrospray ionization mass spectrometry (ESI-MS). Multi-stage tandem mass spectrometric (ESI-MS(n)) data for the alkaloid compounds were used for detailed structural characterization, then structure information was obtained by comparison of the fragmentation mechanisms of both alkaloids in Rhizoma Coptidis and standard samples of berberine, palmatine, coptisine and jatrorrhizine by MS. Based on the results obtained, the structure of a novel compound was elucidated. The results of the experiments demonstrate that ESI-MS(n) is a sensitive, selective and effective tool for the rapid determination of alkaloids in Rhizoma Coptidis.     

Characterization of a novel microperoxidase from Marinobacter hydrocarbonoclasticus by electrospray ionization tandem mass spectrometry

Microperoxidases are small heme-peptides obtained by proteolytic digestion of cytochrome c, exhibiting peroxidase activity. They consist of a short- or medium-length polypeptide chain, covalently linked to an iron protoporphyrin IX moiety via two thioether bonds involving Cys residues at the c-porphyrin A and B pyrrole rings. These small molecules are interesting for a wide range of possible applications. We have structurally characterized, by means of electrospray ionization (ESI) mass and tandem mass spectrometric experiments, a novel microperoxidase called MMP-5 (Marinobacter MicroPeroxidase-5), obtained by proteolytic digestion of cytochrome c552, a monoheminic electron-transfer protein isolated from Marinobacter hydrocarbonoclasticus. This microperoxidase, which still maintains the functional peptide moieties for peroxidase activity, is devoid of the two amino acids intercalating the Cys residues linked to the c-porphyrin, thus increasing its water solubility. Once submitted to the ESI source potential, MMP-5 showed an interesting tendency for the reduction of the iron protoporphyrin substructure. This behaviour was clearly evidenced by the mass shift exhibited by the reduced form.     

Characterization of amino acid‐derived betaines by electrospray ionization tandem mass spectrometry

Betaines belong to the naturally occurring osmoprotectants or compatible solutes present in a variety of plants, animals and microorganisms. In recent years, metabolomic techniques have been emerging as a fundamental tool for biologists because the constellation of these molecules and their relative proportions provide with information about the actual biochemical condition of a biological system. Therefore, identification and characterization of biologically important betaines are crucial, especially for metabolomic studies. Most of the natural betaines are derived from amino acids and related homologues. Although, theoretically, all the amino acids can be converted to corresponding betaines by simple methylation of the amine group, only a few of the amino acid‐derived betaines were fully characterized in the literature. Here, we report a combined electrospray ionization tandem and high‐resolution mass spectrometry study of all the betaines derived from amino acids, including the isomeric betaines. The decomposition pathway of protonated, sodiated and potassiated molecule ions that enable unambiguous characterization of the betaines including the isomeric betaines and overlapping ionic species of different betaines is distinctive. Copyright © 2012 John Wiley & Sons, Ltd.     

Identification and fragmentation of hydrolyzed aluminum species by electrospray ionization tandem mass spectrometry

Earlier characterization of some hydrolysis products of AlCl₃·6H₂O was confirmed by electrospray ionization tandem mass spectrometry with increasing collision energy of projectile ions. At lower collision energies, the aqua ligands were stripped off. At higher energies, two hydroxo groups formed a bridging oxo group with loss of one water molecule. Aluminum complexes could also capture aqua ligands in the collision chamber so long as the parent ion did not fragment, and the fragment ion spectra broadened toward higher m/z values. The chloro ligands were eliminated as hydrochloric acid. The aluminum cores remained highly intact. Copyright © 2004 John Wiley & Sons, Ltd.     

Characterizing the electrospray ionization mass spectral fragmentation pattern of indole derivatives synthesized from 2-keto glycosides

Four indole derivatives synthesized from 2-keto glycosides were analyzed by electrospray ionization tandem mass spectrometry (ESI-MSn) with low-energy collision-induced dissociation to establish a general structural elucidation of indole derivatives. Their fragmentation pathways are proposed on the basis of the MSn studies and deuterium-labeled experiments. Indole derivatives undergo complicated gas-phase rearrangements in addition to simple bond cleavages. A rearrangement, which involves a contraction of the six-membered ring, was observed and a mechanism was proposed. The observations may have some potential applications in the interpretation of the mass spectra of indole derivatives.     

Structural characterization of glycoporphyrins by electrospray tandem mass spectrometry

Porphyrin derivatives having a galactose or a bis(isopropylidene)galactose structural unit, linked by ester or ether bonds, were characterized by electrospray tandem mass spectrometry (ES-MS/MS). The electrospray mass spectra of these glycoporphyrins show the corresponding [M + H](+) ions. For the glycoporphyrins with pyridyl substituents and those having a tetrafluorophenyl spacer, the doubly charged ions [M + 2H](2+) were also observed in ES-MS with high relative abundance. The fragmentation of both [M + H](+) and [M + 2H](2+) ions exhibited common fragmentation pathways for porphyrins with the same sugar residue, independently of the porphyrin structural unit and type of linkage. ES-MS/MS of the [M + H](+) ions of the galactose-substituted porphyrins gave the fragment ions [M + H - C(2)H(4)O(2)](+), [M + H - C(3)H(6)O(3)](+), [M + H - C(4)H(8)O(4)](+) and [M + H - galactose residue](+). The fragmentation of the [M + 2H](2+) ions of the porphyrins with galactose shows the common doubly charged fragment ions [porphyrin + H](2+), [M + 2H - C(2)H(4)O(2)](2+), [M + 2H - C(4)H(8)O(4)](2+), [M + 2H - galactose residue](2+) and the singly charged fragment ions [M + H - C(3)H(6)O(3)](+) and [M + H - galactose residue](+). The fragmentation of the [M + H](+) ions of glycoporphyrins with a protected galactosyl residue leads mainly to the ions [M + H - CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2)](+), [M + H - 2CO(CH(3))(2) - CO](+), [M + H - C(10)H(16)O(4)](+) and [M + H - protected galactose](+). The doubly charged ions [M + 2H](2+) fragment to give the doubly charged ions [porphyrin + H](2+) and the singly charged ions [M + H - protected galactose residue](+) and [M + H - CO(CH(3))(2)](+). For the porphyrins where the sugar structural unit is linked by an ester bond, [M + 2H](2+), ES-MS/MS showed a major and typical fragmentation corresponding to combined loss of a sugar structural unit and further loss of water, leading to the ion [M + 2H - sugar residue - H(2)O](2+), independently of the structure of the sugar structural unit. These results show that ES-MS/MS can be a powerful tool for the characterization of the sugar structural unit of glycoporphyrins, without the need for chemical hydrolysis.     

Selective alkali metal binding of valinomycin by electrospray ionization mass spectometry

Abstract

Selective binding of alkali metal ions by the ionophore antibiotic valinomycin in alcoholic media was detected by electrospray ionization mass spectrometry (ESI-MS). The relative cation affinity for valinomycin is in the order Rb+ > Cs+ > K+ ? Na+ in both methanol and ethanol. With a 1:1:1 mixture of valinomycin and synthetic mimics 18-crown-6 and [2,2,2]-cryptate (methanol, excess KOAc) valinomycin showed much higher affinity than the other ionophores. This study has shown that the potential of ESI-MS as a very useful tool for research involving metal binding is great. Problems with sodium ion interference and quantitation are discussed.     

Novel acetylation-aided migrating rearrangement of uridine-diphosphate-N-acetylglucosamine in electrospray ionization multistage tandem mass spectrometry

Uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) is the final product of hexosamine biosynthetic pathway (HSP) and the donor substrate for the modification of nucleocytoplasmic proteins at serine and threonine residues with N-acetylglucosamine (GlcNAc) catalyzed by O-GlcNAc transferase (OGT). Many analogs of UDP-GlcNAc were designed to interfere with the process of protein O-glycosylation by blocking OGT. A novel rearrangement reaction was observed in which phosphate-N-acetylglucosamine moiety migrated to 3' terminus of ribose in ESI-MS(n) of UDP-GlcNAc. Results from tandem mass spectrometry, control experiments and calculation showed that the phosphate-N-acetylglucosamine migration might undergo a pentacoordinate phosphoric intermediate. Furthermore, the acetylation of glucosamine in UDP-GlcNAc was essential in the migration process.     

Quantitative determination of domperidone in human plasma by ultraperformance liquid chromatography with electrospray ionization tandem mass spectrometry

A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for determining domperidone in human plasma. The analyte and internal standard (IS; mosapride) were isolated from plasma samples by protein precipitation with methanol (containing 0.1% formic acid). The chromatographic separation was performed on an Xterra MS C(18) Column (2.1 x 150 mm, 5.0 microm) with a gradient programme mobile phase consisting of 0.1% formic acid and acetonitrile at a flow rate of 0.30 mL/min. The total run time was 4.0 min. The analyses were carried out by multiple reaction monitoring using the parent-to-daughter combinations m/z 426 --> 175 and m/z 422 --> 198 (IS). The areas of peaks from the analyte and IS were used for quantification of domperidone. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicated that the lower limit of quantification was 0.2 ng/mL, and the assay exhibited a linear range of 0.2-60.0 ng/mL and gave a correlation coefficient (r(2)) of 0.999 or better. Quality control samples (0.4, 0.8, 15 and 50 ng/mL) in six replicates from three different analytical runs demonstrated an intra-assay precision (RSD) 4.43-6.26%, an inter-assay precision 5.25-7.45% and an overall accuracy (relative error) of <6.92%. The method can be applied to pharmacokinetic and bioequivalence studies of domperidone.     

Structural characterization of alachlor complexes with transition metal ions by electrospray ionization tandem mass spectrometry

Electrospray ionization mass (ESI-MS) spectrometry was used to investigate the nature of metal complexes of alachlor and their dissociations on activation. Ions of the first row transition metal series were employed to react with alachlor and the products were subjected to collision-induced dissociation (CID) for further structural characterization. The formation of diverse complex ions including doubly charged metal/alachlor complexes; [3L + M]²⁺ and [4L + M]²⁺ (L: alachlor and M: transition metal ions) were observed depending on the experimental conditions including the tube lens offset voltage (TLOV) and relative concentrations of alachlor and transition metal ions. It is clear that complexation with transition metal ions alters the reactive site of alachlor, promoting the loss of chlorine over the loss of CH₃OH that is the major reaction pathway in uncomplexed system. Direct elimination of chlorine from alachlor molecule was confirmed by the use of MnBr₂ instead of MnCl₂. These evidences clearly illustrate the catalytic activities of the metal ions through insertion mechanism. The function of transition metal ions in complexation was emphasized comparing the fragmentation patterns with those of protonated molecule. A change in the oxidation state of copper from + 2 to + 1 during the dissociation of metal complex was observed in company with elimination of radicals which is specific for the copper complex ions.     

Relationships between structure,ionization profile and sensitivity of exogenous anabolic steroids under electrospray ionization and analysis in human urine using liquid chromatography–tandem mass spectrometry

The relationships between the ionization profile, sensitivity, and structures of 64 exogenous anabolic steroids (groups I–IV) was investigated under electrospray ionization (ESI) conditions. The target analytes were ionized as [M + H]⁺ or [M + H–nH₂O]⁺ in the positive mode, and these ions were used as precursor ions for selected reaction monitoring analysis. The collision energy and Q3 ions were optimized based on the sensitivity and selectivity. The limits of detection (LODs) were 0.05–20 ng/mL for the 64 steroids. The LODs for 38 compounds, 14 compounds and 12 compounds were in the range of 0.05–1, 2–5 and 10–20 ng/mL, respectively. Steroids including the conjugated keto‐functional group at C3 showed good proton affinity and stability, and generated the [M + H]⁺ ion as the most abundant precursor ion. In addition, the LODs of steroids using the [M + H]⁺ ion as the precursor ion were mostly distributed at low concentrations. In contrast, steroids containing conjugated/unconjugated hydroxyl functional groups at C3 generated [M + H ? H₂O]⁺ or [M + H ? 2H₂O]⁺ ions, and these steroids showed relatively high LODs owing to poor stability and multiple ion formation. An LC‐MS/MS method based on the present ionization profile was developed and validated for the determination of 78 steroids (groups I–V) in human urine. Copyright © 2015 John Wiley & Sons, Ltd.     

Differentiation of diiodothyronines using electrospray ionization tandem mass spectrometry

Diiodothyronines 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), and 3,3'-diiodothyronine (3,3'-T2) are important metabolites of 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3; reverse T3). In this paper, a novel and rapid method for identifying and quantifying 3,5-T2, 3',5'-T2 and 3,3'-T2 has been introduced using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed on the basis of our data obtained by ESI-MS/MS. MS2 spectra in either negative ionization mode or positive ionization mode can be used to differentiate 3,5-T2, 3',5'-T2 and 3,3'-T2. On the basis of the relative abundance of fragment ions in MS2 spectra under the positive ionization mode, quantification of the 3,5-T2, 3',5'-T2 and 3,3'-T2 isomers in mixtures is also achieved without prior separation.     

The mass spectral analysis of isolated hops A-type proanthocyanidins by electrospray ionization tandem mass spectrometry

Comprehensive mass spectral fragmentation patterns have been established for sequencing chromatographically isolated A-type proanthocyanidins (PAs) using electrospray ionization tandem mass spectrometry (ESI-MS(n)) in the positive ion mode similar to those used for sequencing previously reported B-type PAs. Sequence-identifying fragmentations for A-type PAs include heterocyclic ring fission (HRF), retro-Diels-Alder (RDA) fission, benzofuran-forming (BFF) fission, and quinone methide (QM) fission. There is commonality in fragmentation patterns between A-type and B-type PAs, but distinguishing features in the mass spectral patterns between the two classes include 2-Da mass differences in the pseudo molecular ions, the propensity for the A-type PAs to undergo QM fissions and yield bis-quinoid ions as opposed to mono-quinoid ions in the upper unit of the sequence, and the reluctance of A-type linkages to undergo RDA, BFF, and BFF/H(2)O fissions from the upper unit. The positions of one or more A-type (C2-->O-->C7') ether linkages have been located in sequences of PAs ranging in chain lengths of two to five monomer units using ESI-MS(n) data. Using the fragmentation information from ESI-MS(n) experiments, a total of 17 PAs were structurally sequenced by systematic real time ESI-MS(n). Among them ten A-type and six B-type hop PAs are reported here for the first time.