Travelling‐wave ion mobility and negative ion fragmentation of high‐mannose N‐glycans

The isomeric structure of high‐mannose N‐glycans can significantly impact biological recognition events. Here, the utility of travelling‐wave ion mobility mass spectrometry for isomer separation of high‐mannose N‐glycans is investigated. Negative ion fragmentation using collision‐induced dissociation gave more informative spectra than positive ion spectra with mass‐different fragment ions characterizing many of the isomers. Isomer separation by ion mobility in both ionization modes was generally limited, with the arrival time distributions (ATD) often showing little sign of isomers. However, isomers could be partially resolved by plotting extracted fragment ATDs of the diagnostic fragment ions from the negative ion spectra, and the fragmentation spectra of the isomers could be extracted by using ions from limited areas of the ATD peak. In some cases, asymmetric ATDs were observed, but no isomers could be detected by fragmentation. In these cases, it was assumed that conformers or anomers were being separated. Collision cross sections of the isomers in positive and negative fragmentation mode were estimated from travelling‐wave ion mobility mass spectrometry data using dextran glycans as calibrant. More complete collision cross section data were achieved in negative ion mode by utilizing the diagnostic fragment ions. Examples of isomer separations are shown for N‐glycans released from the well‐characterized glycoproteins chicken ovalbumin, porcine thyroglobulin and gp120 from the human immunodeficiency virus. In addition to the cross‐sectional data, details of the negative ion collision‐induced dissociation spectra of all resolved isomers are discussed. Copyright © 2016 John Wiley & Sons, Ltd.     

Arrival time distributions of product ions reveal isomeric ratio of deprotonated molecules in ion mobility–mass spectrometry of hyaluronan‐derived oligosaccharides

Hyaluronic acid is a naturally occurring linear polysaccharide with substantial medical potential. In this work, discrimination of tyramine‐based hyaluronan derivatives was accessed by ion mobility–mass spectrometry of deprotonated molecules and nuclear magnetic resonance spectroscopy. As the product ion mass spectra did not allow for direct isomer discrimination in mixture, the reductive labeling of oligosaccharides as well as stable isotope labeling was performed. The ion mobility separation of parent ions together with the characteristic fragmentation for reduced isomers providing unique product ions allowed us to identify isomers present in a mixture and determine their mutual isomeric ratio. The determination used simple recalculation of arrival time distribution areas of unique ions to areas of deprotonated molecules. Mass spectrometry data were confirmed by nuclear magnetic resonance spectroscopy. Copyright © 2015 John Wiley & Sons, Ltd.     

Differentiation of Boc‐protected α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptide positional isomers by electrospray ionization tandem mass spectrometry

Two new series of Boc‐N‐α,δ‐/δ,α‐ and β,δ‐/δ,β‐hybrid peptides containing repeats of L ‐Ala‐δ⁵‐Caa/δ⁵‐Caa‐L ‐Ala and β³‐Caa‐δ⁵‐Caa/δ⁵‐Caa‐β³‐Caa (L ‐Ala = L ‐alanine, Caa = C‐linked carbo amino acid derived from D ‐xylose) have been differentiated by both positive and negative ion electrospray ionization (ESI) ion trap tandem mass spectrometry (MS/MS). MSⁿ spectra of protonated isomeric peptides produce characteristic fragmentation involving the peptide backbone, the Boc‐group, and the side chain. The dipeptide positional isomers are differentiated by the collision‐induced dissociation (CID) of the protonated peptides. The loss of 2‐methylprop‐1‐ene is more pronounced for Boc‐NH‐L ‐Ala‐δ‐Caa‐OCH₃ (1), whereas it is totally absent for its positional isomer Boc‐NH‐δ‐Caa‐L ‐Ala‐OCH₃ (7), instead it shows significant loss of t‐butanol. On the other hand, second isomeric pair shows significant loss of t‐butanol and loss of acetone for Boc‐NH‐δ‐Caa‐β‐Caa‐OCH₃ (18), whereas these are insignificant for its positional isomer Boc‐NH‐β‐Caa‐δ‐Caa‐OCH₃ (13). The tetra‐ and hexapeptide positional isomers also show significant differences in MS² and MS³ CID spectra. It is observed that ‘b’ ions are abundant when oxazolone structures are formed through five‐membered cyclic transition state and cyclization process for larger ‘b’ ions led to its insignificant abundance. However, b₁⁺ ion is formed in case of δ,α‐dipeptide that may have a six‐membered substituted piperidone ion structure. Furthermore, ESI negative ion MS/MS has also been found to be useful for differentiating these isomeric peptide acids. Thus, the results of MS/MS of pairs of di‐, tetra‐, and hexapeptide positional isomers provide peptide sequencing information and distinguish the positional isomers. Copyright © 2010 John Wiley & Sons, Ltd.     

Viscosalines B1,2 and E1,2: Challenging New 3‐Alkyl Pyridinium Alkaloids from the Marine Sponge Haliclona viscosa

Four new 3‐alkyl pyridinium alkaloids, the viscosalines B₁ ( 1 a ), B₂ ( 1 b ), E₁ ( 2 a ), and E₂ ( 2 b ), were isolated from the Arctic sponge Haliclona viscosa. The structure elucidation of these isomeric compounds was challenging due to ambiguous fragments that derive during “standard” mass spectrometric fragmentation experiments. The final structure elucidation relied on the use of a combination of synthesis, liquid chromatography, and mass spectrometry. Three different mass spectrometers were used to differentiate between the synthetic structural isomers: a time‐of‐flight (TOF) mass spectrometer and two ion‐trap mass spectrometers with different ion‐transfer technologies (i.e., skimmer versus funnel optics). Although at first none of the spectrometers returned spectra that permitted structure elucidation, all three mass spectrometers provided analysis that successfully differentiated between the isomers after thorough method optimization. The use of in‐source collision‐induced dissociation (CID) with the ion trap and TOF instrument returned the most interesting results. The mode of fragmentation of the viscosalines under different experimental conditions is described herein. After successful optimization of the mass spectrometric method applied, the chromatographic method was improved to distinguish the previously inseparable isomers. Finally, both the liquid chromatography and mass spectrometric methods were applied to the natural products and the results compared to those from the synthetic compounds.     

Travelling‐wave ion mobility mass spectrometry and negative ion fragmentation of hybrid and complex N‐glycans

Nitrogen collisional cross sections (CCSs) of hybrid and complex glycans released from the glycoproteins IgG, gp120 (from human immunodeficiency virus), ovalbumin, α1‐acid glycoprotein and thyroglobulin were measured with a travelling‐wave ion mobility mass spectrometer using dextran as the calibrant. The utility of this instrument for isomer separation was also investigated. Some isomers, such as Man₃GlcNAc₃ from chicken ovalbumin and Man₃GlcNAc₃Fuc₁ from thyroglobulin could be partially resolved and identified by their negative ion fragmentation spectra obtained by collision‐induced decomposition (CID). Several other larger glycans, however, although existing as isomers, produced only asymmetric rather than separated arrival time distributions (ATDs). Nevertheless, in these cases, isomers could often be detected by plotting extracted fragment ATDs of diagnostic fragment ions from the negative ion CID spectra obtained in the transfer cell of the Waters Synapt mass spectrometer. Coincidence in the drift times of all fragment ions with an asymmetric ATD profile in this work, and in the related earlier paper on high‐mannose glycans, usually suggested that separations were because of conformers or anomers, whereas symmetrical ATDs of fragments showing differences in drift times indicated isomer separation. Although some significant differences in CCSs were found for the smaller isomeric glycans, the differences found for the larger compounds were usually too small to be analytically useful. Possible correlations between CCSs and structural types were also investigated, and it was found that complex glycans tended to have slightly smaller CCSs than high‐mannose glycans of comparable molecular weight. In addition, biantennary glycans containing a core fucose and/or a bisecting GlcNAc residue fell on different mobility‐m/z trend lines to those glycans not so substituted with both of these substituents contributing to larger CCSs. Copyright © 2016 John Wiley & Sons, Ltd.     

Divergent Coordination Chemistry: Parallel Synthesis of [2×2] Iron(II) Grid‐Complex Tauto‐Conformers

The coordination of iron(II) ions by a homoditopic ligand L with two tridentate chelates leads to the tautomerism‐driven emergence of complexity, with isomeric tetramers and trimers as the coordination products. The structures of the two dominant [Fe^II₄ L ₄]⁸⁺ complexes were determined by X‐ray diffraction, and the distinctness of the products was confirmed by ion‐mobility mass spectrometry. Moreover, these two isomers display contrasting magnetic properties (Fe^II spin crossover vs. a blocked Fe^II high‐spin state). These results demonstrate how the coordination of a metal ion to a ligand that can undergo tautomerization can increase, at a higher hierarchical level, complexity, here expressed by the formation of isomeric molecular assemblies with distinct physical properties. Such results are of importance for improving our understanding of the emergence of complexity in chemistry and biology.     

Isomer differentiation via collision‐induced dissociation: The case of protonated α‐, β2‐ and β3‐phenylalanines and their derivatives

A combination of electrospray ionisation (ESI), multistage and high‐resolution mass spectrometry experiments is used to examine the gas‐phase fragmentation reactions of the three isomeric phenylalanine derivatives, α‐phenylalanine, β²‐phenylalanine and β³‐phenylalanine. Under collision‐induced dissociation (CID) conditions, each of the protonated phenylalanine isomers fragmented differently, allowing for differentiation. For example, protonated β³‐phenylalanine fragments almost exclusively via the loss of NH₃, only β²‐phenylalanine via the loss of H₂O, while α‐ and β²‐phenylalanine fragment mainly via the combined losses of H₂O + CO. Density functional theory (DFT) calculations were performed to examine the competition between NH₃ loss and the combined losses of H₂O and CO for each of the protonated phenylalanine isomers. Three potential NH₃ loss pathways were studied: (i) an aryl‐assisted neighbouring group; (ii) 1,2 hydride migration; and (iii) neighbouring group participation by the carboxyl group. Finally, we have shown that isomer differentiation is also possible when CID is performed on the protonated methyl ester and methyl amide derivatives of α‐, β²‐ and β³‐phenylalanines. Copyright © 2010 John Wiley & Sons, Ltd.     

First report of non‐coloured flavonoids in Echium plantagineum bee pollen: differentiation of isomers by liquid chromatography/ion trap mass spectrometry

Apicultural products have been widely used in diet complements as well as in phytotherapy. Bee pollen from Echium plantagineum was analysed by high‐performance liquid chromatography/photodiode‐array detection coupled to ion trap mass spectrometry (HPLC‐PAD‐MSⁿ) with an electrospray ionisation interface. The structures have been determined by the study of the ion mass fragmentation, which characterises the interglycosidic linkage in glycosylated flavonoids and differentiates positional isomers. Twelve non‐coloured flavonoids were characterised, being kaempferol‐3‐O‐neohesperidoside the major compound, besides others in trace amounts. These include quercetin, kaempferol and isorhamnetin glycosides, with several of them being isomers. Acetylated derivatives are also described. This is the first time that non‐coloured flavonoids are reported from this pollen, with MS fragmentation proving to be most useful in the elucidation of isomeric structures. Copyright © 2010 John Wiley & Sons, Ltd.     

New isomeric N‐substituted hydrazones of 2‐, 3‐ and 4‐pyridinecarboxaldehydes and methyl‐3‐pyridylketone

Twelve compounds unknown in the literature N‐(E)‐2‐stilbenyloxymethylenecarbonyl substituted hydrazones of 2‐, 3‐ and 4‐pyridinecarboxaldehydes, as well as methyl‐3‐pyridylketone have been prepared. The stereochemical behavior of these compounds in dimethyl‐d₆ sulfoxide solution has been studied by ¹H NMR technique. The E geometrical isomers and cis/trans amide conformers have been found for N‐substituted hydrazones 1–12. EI induced mass spectral fragmentation of these compounds were also investigated. The data obtained create the basis for distinguishing isomers.     

Structural determination of glycopeptidolipids of Mycobacterium smegmatis by high‐resolution multiple‐stage linear ion‐trap mass spectrometry with electrospray ionization

Glycopeptidolipids (GPLs) are abundant in the cell walls of different species of mycobacteria and consist of tripeptide‐amino‐alcohol core of D‐Phe‐D‐allo‐Thr‐D‐Ala‐L‐alaninol linked to 3‐hydroxy or 3‐methoxy C_26–34 fatty acyl chain at the N‐terminal of D‐Phe via amide linkage, and a 6‐deoxytalose (6‐dTal) and an O‐methyl rhamnose residues, respectively, attach to D‐allo‐Thr and the terminal L‐alaninol. They are important cell‐surface antigens that are implicated in the pathogenesis of opportunistic mycobacteria belonging to the Mycobacterium avium complex. In this contribution, we described multiple‐stage linear ion trap in conjunction with high‐resolution mass spectrometry towards structural characterization of complex GPLs as [M + Na]⁺ ions isolated from Mycobacterium smegmatis, a fast‐growing and non‐pathogenic mycobacterial species. Following resonance excitation in an ion trap, MSⁿ spectra of the [M + Na]⁺ ions of GPLs contained mainly b and y series ions that readily determine the peptide sequence. Fragment ions from MSⁿ also afford locating the 6‐dTal and O‐methyl rhamnose residues linked to the D‐allo‐Thr and terminal L‐alaninol of the peptide core, respectively, as well as recognizing the modifications of the glycosides, including their acetylation and methylation states and the presence of succinyl group. The GPL families consisting of 3‐hydroxy fatty acyl and of 3‐methoxy fatty acyl substituents are readily distinguishable. The MS profiles of the GPLs from cells are dependant on the conditions they were grown, and several isobaric isomers were identified for many of the molecular species. These multiple‐stage mass spectrometric approaches give detailed structures of GPL in complex mixtures of which the isomeric structures are difficult to define using other analytical methods. Copyright © 2012 John Wiley & Sons, Ltd.     

Structural motifs in primary oxidation products of palmitoyl‐arachidonoyl‐phosphatidylcholines by LC‐MS/MS

Oxidative modifications to phospholipids (OxPL) play a major role in modulating signaling events in inflammation and infection, and complete understanding on the induced biological effects can only be understood based on knowledge of the oxidative motifs present. Specific neutral losses observed in tandem mass spectrometry data (LC‐MS/MS) of primary peroxidation products in oxidized palmitoyl‐arachidonoyl‐phosphatidylcholines (OxPAPC) provide information on the prevailing structural motifs regarding the oxidized acyl carbon chain, the nature of oxidized group and the site of carbon oxidation. The higher hydrophobicity of hydroperoxides compared to di‐hydroxy derivatives under reverse‐phase conditions together with specific fragmentation patterns enabled the identification of 12 structurally different OxPAPC structural (di‐hydroxy and hydroperoxide derivatives) and positional isomers as well as the presence of poly‐hydroxy together with isoprostanes derivatives. The fragmentation patterns described in quadrupole time‐of‐flight and linear ion trap instruments complement the m/z value and retention time parameters in the identification of oxidative composition in OxPAPC products becoming a valuable tool for the exploratory screening of oxidized phosphatidylcholines in OxPAPC extracts, distinction of native and modified PC isobaric structures in complex samples contributing to the increased understanding of redox lipidomics in inflammation and infection. Copyright © 2013 John Wiley & Sons, Ltd.     

Structural characterization of the ligstroside aglycone isoforms in virgin olive oils by liquid chromatography–high‐resolution Fourier‐transform mass spectrometry and H/Dexchange

A systematic structural characterization of the isomeric forms related to ligstroside aglycone (LA), one of the most relevant secoiridoids contained in virgin olive oils, was performed using reverse phase liquid chromatography with electrospray ionization Fourier‐transform single and tandem mass spectrometry, operated in negative ion mode (RPLC‐ESI(?)‐FTMS and FTMS/MS). The high mass resolution and accuracy provided by the adopted orbital trap mass analyzer enabled the recognition of more than 10 different isomeric forms of LA in virgin olive oil extracts. They were related to four different types of molecular structure, two of which including a dihydropyranic ring bearing one or two aldehydic groups, whereas the others corresponded to dialdehydic open‐structure forms, differing just for the position of a C═C bond. The contemporary presence of enolic or dienolic tautomers associated to most of these compounds, stable at room temperature (23°C), was also assessed through RPLC‐ESI‐FTMS analyses operated under H/D exchange conditions, ie, by using D₂O instead of H₂O as co‐solvent of acetonitrile in the RPLC mobile phase. As discussed in the paper, the results obtained for LA indicated a remarkable structural similarity with oleuropein aglycone (OA), the most abundant secoiridoid of olive oil, whose isoforms had been previously characterized using the same analytical approach.     

Peak width‐mass correlation in CID MS/MS of isomeric oligosaccharides using traveling‐wave ion mobility mass spectrometry

Isomeric oligosaccharides γ‐cyclodextrin (γ‐CD), glucosyl‐βCD (Glc₁‐βCD) and maltosyl‐αCD (Glc₂‐αCD) were analyzed by traveling‐wave ion mobility (twIM) mass spectrometry (MS). Their formation of multicharged multimers differed from each other. The ion mobility‐mass spectrometry was useful in the self‐assembling and complex formation analyses of CD isomers. The drift times of the isomers and their product ions with the same mass were almost the same in collision‐induced dissociation (CID) MS/MS. In contrast, the ion mobility peak widths were sensitive to structural differences of the isomeric product ions. The twIM peak width (ms ‐ µs) of the product ions [M ? Glc_n + H]⁺ (n = 0 ～ 6) of γ‐CD correlated linearly with their masses (Da); the large and/or long chain product ions had wider peak widths, which were much wider than those from the general diffusion effect. This was a novel and useful ‘trend line’ to discriminate between the three isomers. Plots of [M ? Glc_{2 ～ 6} + H]⁺ of Glc₁‐βCD and [M ? Glc_{3 ～ 6} + H]⁺ of Glc₂‐αCD product ions' plots were on the same trend line as γ‐CD. The plots of [M ? Glc₁ + H]⁺ of Glc₁‐βCD and [M ? Glc_{1, 2} + H]⁺ of Glc₂‐αCD strayed from the γ‐CD line; their peak widths were narrower than those of γ‐CD. These results indicated that product ions from the chemical species of Glc₁‐β CD and Glc₂‐αCD retained their CD structure. Analyses of the IM peak widths enable us to elucidate the structures of the product ions. Copyright © 2009 John Wiley & Sons, Ltd.     

Differentiation of isomeric dinitrotoluenes and aminodinitrotoluenes using electrospray high resolution mass spectrometry

Explosive detection and identification play an important role in the environmental and forensic sciences. However, accurate identification of isomeric compounds remains a challenging task for current analytical methods. The combination of electrospray multistage mass spectrometry (ESI‐MSⁿ) and high resolution mass spectrometry (HRMS) is a powerful tool for the structure characterization of isomeric compounds. We show herein that resonant ion activation performed in a linear quadrupole ion trap allows the differentiation of dinitrotoluene isomers as well as aminodinitrotoluene isomers. The explosive‐related compounds: 2,4‐dinitrotoluene (2,4‐DNT), 2,6‐dinitrotoluene (2,6‐DNT), 2‐amino‐4,6‐dinitrotoluene (2A‐4,6‐DNT) and 4‐amino‐2,6‐dinitrotoluene (4A‐2,6‐DNT) were analyzed by ESI‐MS in the negative ion mode; they produced mainly deprotonated molecules [M ? H]^?. Subsequent low resolution MSⁿ experiments provided support for fragment ion assignments and determination of consecutive dissociation pathways. Resonant activation of deprotonated dinitrotoluene isomers gave different fragment ions according to the position of the nitro and amino groups on the toluene backbone. Fragment ion identification was bolstered by accurate mass measurements performed using Fourier transform ion cyclotron resonance mass spectrometry (FT‐ICR/MS). Notably, unexpected results were found from accurate mass measurements performed at high resolution for 2,6‐DNT where a 30‐Da loss was observed that corresponds to CH₂O departure instead of the expected isobaric NO^? loss. Moreover, 2,4‐DNT showed a diagnostic fragment ion at m/z 116, allowing the unambiguous distinction between 2,4‐ and 2,6‐DNT isomers. Here, CH₂O loss is hindered by the presence of an amino group in both 2A‐4,6‐DNT and 4A‐2,6‐DNT isomers, but nevertheless, these isomers showed significant differences in their fragmentation sequences, thus allowing their differentiation. DFT calculations were also performed to support experimental observations. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous qualitative and quantitative analysis of flavonoids and alkaloids from the leaves of Nelumbo nucifera Gaertn. using high‐performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

A reliable method, combining qualitative analysis by high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry and quantitative assessment by high‐performance liquid chromatography with photodiode array detection, has been developed to simultaneously analyze flavonoids and alkaloids in lotus leaf extracts. In the qualitative analysis, a total of 30 compounds, including 12 flavonoids, 16 alkaloids, and two proanthocyanidins, were identified. The fragmentation behaviors of four types of flavone glycoside and three types of alkaloid are summarized. The mass spectra of four representative components, quercetin 3‐O‐glucuronide, norcoclaurine, nuciferine, and neferine, are shown to illustrate their fragmentation pathways. Five pairs of isomers were detected and three of them were distinguished by comparing the elution order with reference substances and the mass spectrometry data with reported data. In the quantitative analysis, 30 lotus leaf samples from different regions were analyzed to investigate the proportion of eight representative compounds. Quercetin 3‐O‐glucuronide was found to be the predominant constituent of lotus leaf extracts. For further discrimination among the samples, hierarchical cluster analysis, and principal component analysis, based on the areas of the eight quantitative peaks, were carried out.     

Development of a mass spectrometric hydroxyl‐position determination method for the hydroxyindole metabolites of JWH‐018 by GC‐MS/MS

One of the many issues of designer drugs of abuse like synthetic cannabinoids (SCs) such as JWH‐018 is that details on their metabolism has yet to be fully elucidated and that multiple metabolites exist. The presence of isomeric compounds poses further challenges in their identification. Our group has previously shown the effectiveness of gas chromatography‐electron ionization‐tandem mass spectrometry (GC‐EI‐MS/MS) in the mass spectrometric differentiation of the positional isomers of the naphthoylindole‐type SC JWH‐081, and speculated that the same approach could be used for the metabolite isomers. Using JWH‐018 as a model SC, the aim of this study was to differentiate the positional isomers of its hydroxyindole metabolites by GC‐MS/MS. Standard compounds of JWH‐018 and its hydroxyindole metabolite positional isomers were first analyzed by GC‐EI‐MS in full scan mode, which was only able to differentiate the 4‐hydroxyindole isomer. Further GC‐MS/MS analysis was performed by selecting m/z 302 as the precursor ion. All four isomers produced characteristic product ions that enabled the differentiation between them. Using these ions, MRM analysis was performed on the urine of JWH‐018 administered mice and determined the hydroxyl positions to be at the 6‐position on the indole ring. GC‐EI‐MS/MS allowed for the regioisomeric differentiation of the hydroxyindole metabolite isomers of JWH‐018. Furthermore, analysis of the fragmentation patterns suggests that the present method has high potential to be extended to hydroxyindole metabolites of other naphthoylindole type SCs in identifying the position of the hydroxyl group on the indole ring. Copyright © 2016 John Wiley & Sons, Ltd.     

Synthesis and structural characterization of isomeric coumarino‐1,2‐oxaphosphole derivatives

The coumarino‐[3,4‐c]‐3H‐10‐methyl‐2‐oxo‐2‐phenyl‐1,2‐oxaphosphole was prepared by the addition of PhPCl₂ to 3‐acetylcoumarin in the presence of acetic anhydride. Its conversion to the isomeric coumarino‐[3,4‐c]‐9H‐9‐methyl‐2‐oxo‐2‐phenyl‐1,2‐oxaphosphole was studied in different reaction conditions. The structures of the two isomers were determined by X‐ray crystallography and by ab initio molecular orbital calculations.     

Fragmentation pathways and differentiation of positional isomers of sorafenib and structural analogues by ESI‐IT‐MSn and ESI‐Q‐TOF‐MS/MS coupled with DFT calculations

Sorafenib is an orally active multikinase inhibitor for the treatment of renal cell carcinoma. A series of sorafenib structural analogues were investigated in this work for their gas‐phase fragmentation behaviors using electrospray ionization ion trap mass spectrometry and quadrupole time‐of‐flight mass spectrometry in the positive mode. The possible fragmentation pathways were proposed based on ESI‐MS/MS data and theoretical calculation. Different from the typical α‐cleavage of amide, consecutive reactions that involved elimination of H₂O and CH₃NC were observed for 2‐pyridinecarboxamide derivatives, which were followed by the formation of a stabilized 7‐membered ring carbocation by loss of CO. Two possible protonation sites occurred at carbonyl oxygen atoms for aryl‐urea derivatives and the α‐cleavage of urea was the main fragmentation pathways, which was followed by the formation of stable benzo [d] oxazole ring characteristic to aryl‐urea derivatives. The relative abundance of characteristic fragment ions and the energy‐resolved breakdown curves were used to distinguish the 4 sets of positional isomers of sorafenib and analogues. The methodology and results of the present work would contribute to the chemical structure identification of other structural analogues and the potential impurities presented in active pharmaceutical ingredients and drug formulations.     

Characterization of superoxide dismutase 1 (SOD‐1) by electrospray ionization‐ion mobility mass spectrometry

In this paper, we report nano‐electrospray ionization‐ion mobility mass spectrometry (nano‐ESI‐IM‐MS) characterization of bovine superoxide dismutase (SOD‐1) and human SOD‐1 purified from erythrocytes. SOD‐1 aggregates are characteristic of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease in humans that could be triggered by dissociation of the native dimeric enzyme (Cu₂,Zn₂‐dimer SOD‐1). In contrast to ESI‐MS, nano‐ESI‐IM‐MS allowed an extra dimension for ion separation, yielding three‐way mass spectra (drift time, mass‐to‐charge ratio and intensity). Drift time provided valuable structural information related to ion size, which proved useful to differentiate between the dimeric and monomeric forms of SOD‐1 under non denaturing conditions. In order to obtain detailed structural information, including the most relevant post‐translational modifications, we evaluated several parameters of the IM method, such as sample composition (10 mM ammonium acetate, pH 7) and activation voltages (trap collision energy and cone voltage). Neutral pH and a careful selection of the most appropriate activation voltages were necessary to minimize dimer dissociation, although human enzyme resulted less prone to dissociation. Under optimum conditions, a comparison between monomer‐to‐dimer abundance ratios of two small sets of blood samples from healthy control and ALS patients demonstrated the presence of a higher relative abundance of Cu,Zn‐monomer SOD‐1 in patient samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Acid‐based approach for separation of peptide epimers using IM‐MS

Chiral molecules frequently remain undistinguishable using ion mobility mass spectrometry (IM‐MS), due to insufficient differences of their collision cross sections at the available mobility resolution of the ion mobility drift tubes. The influence of the complexation with organic acids on the ion mobility separation of peptide epimers is evaluated using traveling‐wave ion mobility (TWIMS). The examined epimeric tripeptides containing Arg residue with the sequence: Ac‐Phe‐Arg‐Trp‐NH₂ formed stable complexes in the gas phase, and under the increased pressure in ion mobility drift tube, noncovalent associates formed with carboxylic or sulfonic monoacids and diacids with chiral variation of certain acids. Overall, the complexation with an acid leads to the improvement in stereodifferentiation among epimeric peptides, in comparison to the analysis of pure epimers. Detailed characterization of peptide epimer‐acid associates obtained for dibenzoyl‐D‐tartaric acid by theoretical calculations and collisional dissociation studies revealed that the presence of multiple hydrogen bonding interactions between carboxylate anions and hydrogens from N―H of both the guanidinium group of arginine and the indole of tryptophan, as well as the amide backbone hydrogens in the peptide, is responsible for stability of acid‐peptide complexes and for their differentiation in the ion mobility drift tube. The specificity of complex formation toward Arg was determined in terms of complex stability. Based on the reported results, we present general conclusions regarding the utility of the acid‐based complexation in the separation of peptide isomers.