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The inability of current mass spectrometry techniques to differentiate phospholipid isomers results in a routine under-estimation of phospholipid molecular diversity in complex biological matrices. Recent technological advances in tandem mass spectrometry and ion activation are helping to overcome these limitations, but all rely on tandem mass spectrometry with unit mass-selection and suffer from co-isolation of isobaric or isomeric species. Accordingly, separation of phospholipid isomers and isobars prior to characterization is required to fully delve into the complexity of the lipidome. Here we present a novel two-stage workflow combining reversed-phase ultra-high performance liquid chromatography with ozone-induced dissociation (OzID) and combined-collision- and ozone-induced-dissociation (COzID) that reduces spectral complexity and enables discrimination of lipid isomers and isobars. Application of this technique to the analysis of human red blood cell lipid extracts allowed the separation, or partial separation, of adduct ion and head group isobars as well as double bond and sn-positional isomers affording near complete structural characterization of low abundance lipids, e.g. PC 18:0/20:3(n-6), PS 18:0/20:4(n-6) and PS 20:4(n-6)/18:0 all observed at m/z 834.7. We also introduce a software plug-in that automatically annotates OzID mass spectra to assign the carbon–carbon double bond positions in lipids. This new workflow allows us to delve deeper into the lipidome and represents another valuable tool for the lipidomics toolbox.

Double bond position and stereochemistry in unsaturated lipids can have profound impact on biological properties and activities but the assignment of these features by mass spectrometry is frequently challenging. Conventional techniques for lipid identification rely on collision-induced dissociation (CID) and are most often unable to differentiate between lipid isomers, particularly those involving double bond position and geometry (i.e., cis and trans). In this study, CID performed on proton-bound complexes of fatty acid methyl esters and iodoaniline (and related reagents) reveals unusual fragmentation patterns. CID products are shown to result from proton transfer and are associated with specific structures of the unsaturated lipids. Notably, CID of these complexes can not only distinguish cis- and trans-fatty acid methyl esters, but also differentiate conjugated double bond arrangements from non-conjugated analogs. Herein, the mechanisms underpinning this unique CID behavior are investigated by stable isotope labeling and are proposed to involve both carbene and free radical intermediates.

The extensive use of alkoxyamines in controlled radical polymerisation and polymer stabilisation is based on rapid cycling between the alkoxyamine (R1R2NO R3) and a stable nitroxyl radical (R1R2NO•) via homolysis of the labile O C bond. Competing homolysis of the alkoxyamine N O bond has been predicted to occur for some substituents leading to production of aminyl and alkoxyl radicals. This intrinsic competition between the O C and N O bond homolysis processes has to this point been difficult to probe experimentally. Herein we examine the effect of local molecular structure on the competition between N O and O C bond cleavage in the gas phase by variable energy tandem mass spectrometry in a triple quadrupole mass spectrometer. A suite of cyclic alkoxyamines with remote carboxylic acid moieties (HOOC R1R2NO R3) were synthesised and subjected to negative ion electrospray ionisation to yield [M − H]− anions where the charge is remote from the alkoxyamine moiety. Collision-induced dissociation of these anions yield product ions resulting, almost exclusively, from homolysis of O C and/or N O bonds. The relative efficacy of N O and O C bond homolysis was examined for alkoxyamines incorporating different R3 substituents by varying the potential difference applied to the collision cell, and comparing dissociation thresholds of each product ion channel. For most R3 substituents, product ions from homolysis of the O C bond are observed and product ions resulting from cleavage of the N O bond are minor or absent. A limited number of examples were encountered however, where N O homolysis is a competitive dissociation pathway because the O C bond is stabilised by adjacent heteroatom(s) (e.g. R3 = CH2F). The dissociation threshold energies were compared for different alkoxyamine substituents (R3) and the relative ordering of these experimentally determined energies is shown to correlate with the bond dissociation free energies, calculated by ab initio methods. Understanding the structure-dependent relationship between these rival processes will assist in the design and selection of alkoxyamine motifs that selectively promote the desirable O C homolysis pathway.

Much of what we currently understand about the structure and energetics of multiply charged anions in the gas phase is derived from the measurement of photoelectron spectra of simple dicarboxylate dianions. Here we have employed a modified linear ion-trap mass spectrometer to undertake complementary investigations of the ionic products resulting from laser-initiated electron photodetachment of two model dianions. Electron photodetachment (ePD) of the [M−2H]2− dianions formed from glutaric and adipic acid were found to result in a significant loss of ion signal overall, which is consistent with photoelectron studies that report the emission of slow secondary electrons (Xing et al., 2010 [20] ). The ePD mass spectra reveal no signals corresponding to the intact [M−2H] − radical anions, but rather [M−2H−CO2] − ions are identified as the only abundant ionic products indicating that spontaneous decarboxylation follows ejection of the first electron. Interestingly however, investigations of the structure and energetics of the [M−2H−CO2] − photoproducts by ion-molecule reaction and electronic structure calculation indicate that (i) these ions are stable with respect to secondary electron detachment and (ii) most of the ion population retains a distonic radical anion structure where the radical remains localised at the position of the departed carboxylate moiety. These observations lead to the conclusion that the mechanism for loss of ion signal involves unimolecular rearrangement reactions of the nascent [M−2H] − carbonyloxyl radical anions that compete favourably with direct decarboxylation. Several possible rearrangement pathways that facilitate electron detachment from the radical anion are identified and are computed to be energetically accessible. Such pathways provide an explanation for prior observations of slow secondary electron features in the photoelectron spectra of the same dicaboxylate dianions.

Results of mass spectrometric studies are reported for the collisional dissociation of Group XI (Cu, Ag, Au) metal ion complexes with fatty acids (palmitic, oleic, linoleic and a-linolenic) and glycerolipids. Remarkably, the formation of M2H+ ions (M = Cu, Ag) is observed as a dissociation product of the ion complexes containing more than one metal cation and only if the lipid in the complex contains a double bond. Ag2H+ is formed as the main dissociation channel for all three of the fatty acids containing double bonds that were investigated while Cu2H+ is formed with one of the fatty acids and, although abundant, is not the dominant dissociation channel. Also. Cu(I) and Ag(I) ion complexes were observed with glycerolipids (including triacylglycerols and glycerophospholipids) containing either saturated or unsaturated fatty acid substituents. Interestingly. Ag2H+ ion is formed in a major fragmentation channel with the lipids that are able to form the complex with two metal cations (triacylglycerols and glycerophosphoglycerols), while lipids containing a fixed positive charge (glycerophospocholines) complex only with a single metal cation. The formation of Ag2H+ ion is a significant dissociation channel from the complex ion Ag-2(L-H)(+) where L = Glycerophospholipid (GP) (18:1/18:1). Cu(I) also forms complexes of two metal cations with glycerophospholipids but these do not produce Cu2H+ upon dissociation. Rather organic fragments, not containing Cu(I), are formed, perhaps due to different interactions of these metal cations with lipids resulting from the much smaller ionic radius of Cu(I) compared to Ag(I) (C).

The isomeric dicoordinated borinium ions CH3O–B–H+ and CH3–B–OH+ are generated upon electron ionization of trimethylborate and methyl boronic acid, respectively. The connectivity of the ions is confirmed by collision-induced dissociation experiments on magnetic deflection type tandem mass spectrometers. Neutralization–reionization experiments on these structurally characterized ions indicate that the neutral radicals CH3O–B–H· and CH3–B–OH· are viable species in the gas phase. Calculations at the G2 level of theory were performed to obtain thermochemical data on the title isomers and their main dissociation products. The calculations also provide a rationale for the moderate yield of the neutrals generated in the experiments: the vertical electron transfer processes for both systems are associated with particularly unfavourable Franck-Condon factors.

The dicoordinated borinium ion, dihydroxyborinium, B(OH) 2 + is generated from methyl boronic acid CH 3 B(OH) 2 by dissociative electron ionization and its connectivity confirmed by collisional activation. Neutralization–reionization (NR) experiments on this ion indicate that the neutral B(OH) 2 radical is a viable species in the gas phase. Both vertical neutralization of B(OH) 2 + and reionization of B(OH) 2 in the NR experiment are, however, associated with particularly unfavorable Franck-Condon factors. The differences in adiabatic and vertical electron transfer behavior can be traced back to a particular π stabilization of the cationic species compared to the sp 2 -type neutral radical. Thermochemical data on several neutral and cationic boron compounds are presented based on calculations performed at the G2 level of theory.

Capture of an electron by tetracyanoethylene oxide can initiate a number of decomposition pathways. One of these decompositions yields [(NC)3C]− as the ionic product. Ab initio calculations (at the B3LYP/6-31+G∗ level of theory) indicate that the formation of [(NC)3C]− is initiated by capture of an electron into the LUMO of tetracyanoethylene oxide to yield the anion radical [(NC)2C–O–C(CN)2]−· that undergoes internal nucleophilic substitution to form intermediate [(NC)3C–OCCN]−·. This intermediate dissociates to form [(NC)3C]− (m/z 90) as the ionic product. The radical (NC)3C· has an electron affinity of 4.0 eV (385 kJ mol−1). Ab initio calculations show that [(NC)3C]− is trigonal planar with the negative charge mainly on the nitrogens. A pictorial representation of this structure is the resonance structure formed from three degenerate contributing structures (NC)2–CCN−. The other product of the reaction is nominally (NCCO)·, but there is no definitive experimental evidence to indicate whether this radical survives intact, or decomposes to NC· and CO. The overall process [(NC)2C–O–C(CN)2]−· → [(NC)3C]− + (NCCO)· is calculated to be endothermic by 21 kJ mol−1 with an overall barrier of 268 kJ mol−1.

Both [C4CO]−· and [C2COC2]−· are formed in the ion source of a VG ZAB 2HF mass spectrometer by the respective processes HO− + Me3Si–CC–CC–CO–CMe3 → [C4CO]−· + Me3SiOH + Me3C·, and Me3Si–CC–CO–CC–SiMe3 + SF6 + e → [C2COC2]−· + 2Me3SiF + SF4. The second synthetic pathway involves a double desilylation reaction similar to that first reported by Squires. The two radical anion isomers produce different and characteristic charge reversal spectra upon collisional activation. In contrast, following collision induced charge stripping, both radical anions produce neutral C4CO as evidenced by the identical neutralisation reionisation (−NR+) spectra. The exclusive rearrangement of C213COC2 to C413CO indicates that 12C–O bond formation is not involved in the reaction. Ab initio calculations (at the RCCSD(T)/aug-cc-pVDZ//B3LYP/6-31G∗ level of theory) have been used to investigate the reaction coordinates on the potential surfaces for both singlet and triplet rearrangements of neutral C2COC2. Singlet C2COC2 is less stable than singlet C4CO by 78.8 kcal mol−1 and requires only 8.5 kcal mol−1 of additional energy to effect conversion to C4CO by a rearrangement sequence involving three C–C ring opening/cyclisation steps.

The gas phase structures of the [M–H] cations and anions of glycine have been studied by using a combination of ab initio calculations (at the MP2(FC)/6–31+G∗ level of theory) and tandem mass spectrometry (MS/MS). It was found that the ab initio stability order for the anions is [H 2 NCH 2 CO 2 ] − > [H 2 NCHCO 2 H] − > [HNCH 2 CO 2 H] − . In contrast, the cations exhibit different behaviour, whereas [H 2 NCHCO 2 H] + is predicted to be a stable structure, [H 2 NCH 2 CO 2 ] + spontaneously fragments to the ion–molecule complex [H 2 NCH 2 + ⋯ (OCO)] and the singlet [HNCH 2 CO 2 H] + isomer is predicted to undergo a skeletal rearrangement to form [CH 2 NHCO 2 H] + . MS/MS spectra of [M–H] + cations of various glycine isotopomers were obtained via: (i) collisional activation of electron impact generated cations and (ii) charge reversal of anions formed via HO − negative ion chemical ionization. The resulting spectra were significantly different, suggesting different structures were involved. Neutralization–reionization experiments were performed on [M–H] − anions in order to gain insights into the structures of the intermediate radicals.