An isomer-specific high-energy collision-induced dissociation MS/MS database for forensic applications: a proof-of-concept on chemical warfare agent markers

Spectra database search has become the most popular technique for the identification of unknown chemicals, minimizing the need for authentic reference chemicals. In the present study, an isomer‐specific high‐energy collision‐induced dissociation (CID) MS/MS spectra database of 12 isomeric O‐hexyl methylphosphonic acids (degradation markers of nerve agents) was created. Phosphonate anions were produced by the electrospray ionization of phosphonic acids or negative‐ion chemical ionization of their fluorinated derivatives and were analysed in a hybrid magnetic‐sector–time‐of‐flight tandem mass spectrometer. A centre‐of‐mass energy (E_com) of 65 eV led to an optimal sequential carbon–carbon bond breakage, which was interpreted in terms of charge remote fragmentation. The proposed mechanism is discussed in comparison with the routinely used low‐energy CID MS/MS. Even‐mass (odd‐electron) charge remote fragmentation ion series were diagnostic of the O‐alkyl chain structure and can be used to interpret unknown spectra. Together with the odd‐mass ion series, they formed highly reproducible, isomer‐specific spectra that gave significantly higher database matches and probability factors (by 1.5 times) than did the EI MS spectra of the trimethylsilyl derivatives of the same isomers. In addition, ionization by negative‐ion chemical ionization and electrospray ionization resulted in similar spectra, which further highlights the general potential of the high‐energy CID MS/MS technique. Copyright © 2011 John Wiley & Sons, Ltd.     

Understanding the Charge Issues in Mono and di-Quaternary Ammonium Compounds for Their Determination by LC/ESI-MS/MS

Chromatographers often develop problems while optimizing a method for the quantification of quaternary ammonium compounds in ESI-MS/MS. Intransigency observed in quaternary ammonium compounds to undergo the classical molecular adduct formation [M+H]⁺ in ESI-MS/MS reduces confidence among chromatographers while working with unit mass resolution. In this study, we provide the evidence for an exceptional rule followed by mono- and di-quaternary ammonium compounds in ESI-MS/MS in the precursor ion formation. Under ESI conditions mono- and di-quaternary ammonium compounds form molecular ions with the formula of m ^q / z ^q rather than ( m + z )/ z . Formation of m ^q / 2 is observed for di-quaternary ammonium compounds in precursor ion scan and m ^q / 1 in product ion scan, if loss of one of the quaternary charge occurs during CID. In di-quaternary ammonium compounds, this process can also result in the formation of fragment ions with higher mass as compared to precursor ion. Hydrophilic interaction liquid chromatographic separation has been used to demonstrate the elution of quaternary ammonium compounds in a single run in the ESI-MS/MS. This work concludes that the analyst must realize and consider these charge issues while dealing with positively charged compounds in LC/ESI-MS/MS.     

Food contaminant analysis at ultra‐high mass resolution: application of hybrid linear ion trap – orbitrap mass spectrometry for the determination of the polyether toxins,azaspiracids, in shellfish

The biotoxins, azaspiracids (AZAs), from marine phytoplankton accumulate in shellfish and affect human health by causing severe gastrointestinal disturbance, diarrhea, nausea and vomiting. Specific and sensitive methods have been developed and validated for the determination of the most commonly occurring azaspiracid analogs. An LTQ Orbitrap mass spectrometer is a hybrid instrument that combines linear ion trap (LIT) mass spectrometry (MS) with high‐resolution Fourier transform (FT) MS and this was exploited to perform simultaneous ultra‐high‐resolution full‐scan MS analysis and collision‐induced dissociation (CID) tandem mass spectrometry (MS/MS). Using the highest mass resolution setting (100 000 FWHM) in full‐scan mode, the methodology was validated for the determination of six AZAs in mussel (Mytilus galloprovincialis) tissue extracts. Ultra‐high mass resolution, together with a narrow mass tolerance window of ±2 mDa, dramatically improved detection sensitivity. In addition to employing chromatographic resolution to distinguish between the isomeric azaspiracid analogs, AZA1/AZA6 and AZA4/AZA5, higher energy collisionally induced dissociation (HCD) fragmentation on selected precursor ions were performed in parallel with full‐scan FTMS. Using HCD MS/MS, most precursor and product ion masses were determined within 1 ppm of the theoretical m/z values throughout the mass spectral range and this enhanced the reliability of analyte identity. For the analysis of mussels (M. galloprovincialis), the method limit of quantitation (LOQ) was 0.010 µg/g using full‐scan FTMS and this was comparable with the LOQ (0.007 µg/g) using CID MS/MS. The repeatability data were; intra‐day RSD% (1.8–4.4%; n = 6) and inter‐day RSD% (4.7–8.6%; n = 3). Application of these methods to the analysis of mussels (M. edulis) that were naturally contaminated with azaspiracids, using high‐resolution full‐scan Orbitrap MS and low‐resolution CID MS/MS, produced equivalent quantitative data. Copyright © 2010 John Wiley & Sons, Ltd.     

Molecular formula analysis of fragment ions by isotope‐selective collision‐induced dissociation tandem mass spectrometry of pharmacologically active compounds

The purpose of this work is to explore the mass fragment characterization of commonly used drugs through a novel approach, which involves isotope‐selective tandem mass spectrometry (MS/MS). Collision‐induced dissociation (CID) was performed with a low‐resolution linear ion trap mass spectrometer in positive electrospray ionization. Three pharmacologically active ingredients, i.e. omeprazole, meloxicam and brinzolamide, selected as model compounds in their own formulation, were investigated as a sodiated adduct [C₁₇H₁₉N₃O₃S + Na]⁺ (omeprazole) and as protonated adducts, [C₁₄H₁₃N₃O₄S₂ + H]⁺ and [C₁₂H₂₁N₃O₅S₃ + H]⁺, meloxicam and brinzolamide, respectively. Selecting a narrow window of ±0.5 m/z units, precursor ion fragmentation by CID‐MS/MS of isotopologues A + 0, A + 1 and A + 2 was found very useful to confirm the chemical formula of product ions, thus aiding the establishment of characteristic fragmentation pathways of all three examined compounds. The correctness of putative molecular formula of product ions was easily demonstrated by exploiting the isotope peak abundance ratios (i.e. I_F+0/I_F+1 and I_F+0/I_F+2) as simple constraints in low‐resolution MS instrumentations. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of a tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source

A new tandem time‐of‐flight mass spectrometer with an electrospray ionization ion source ‘ESI‐TOF/quadTOF’ was designed and constructed to achieve the desired aim of structural elucidation via high‐energy collision‐induced dissociation (CID), and the simultaneous detection of all fragment ions. The instrument consists of an orthogonal acceleration‐type ESI ion source, a linear TOF mass spectrometer, a collision cell, a quadratic‐field ion mirror and a microchannel plate detector. High‐energy CID spectra of doubly protonated angiotensin II and bradykinin were obtained. Several fragment ions such as a‐, d‐, v‐ and w‐type ions, characteristic of high‐energy CID, were clearly observed in these spectra. These high‐energy CID fragment ions enabled confirmation of the complete sequence, including leucine–isoleucine determinations. It was demonstrated that high‐energy CID of multiply protonated peptides could be achieved in the ESI‐TOF/quadTOF. Copyright © 2010 John Wiley & Sons, Ltd.     

High-resolution excitation of ions in a low-pressure linear ion trap

An exploration of the parameters necessary to obtain high‐resolution excitation, using dipolar excitation, of an ion in a linear ion trap has been undertaken in this study. These parameters included ion trap pressure, excitation amplitude, excitation period, drive frequency of the ion trap, Mathieu q value and the mass of the ion of interest. An understanding of how these parameters play a role in high‐resolution excitation is necessary to the development of a method for the targeted tandem mass spectrometric (MS/MS) analysis of ions with the same nominal mass. Resonance excitation profiles with full width half maxima as narrow as 0.015 m/z units could be obtained, under the right conditions, for an ion from a homogenously substituted triazatriphosphorine at m/z 322.049, which translates into a mass resolution of >21 500. In this particular case the requirement for high resolution was a low trap pressure (3.8 × 10^?5 Torr), low excitation amplitude (3 mV), long excitation period (100 ms) and a high Mathieu q value(0.8) when using a drive frequency of 1.228 MHz. Similar conditions were used to demonstrate the isolation of individual [M + H]⁺ component ions from mixtures of bromazepam (m/z 316.008)/chlorprothixene (m/z 316.0921)/fendiline (m/z 316.206) and chlorprothixene (m/z 316.0921)/oxycodone (m/z 316.1543)/fendiline (m/z 316.206) prior to obtaining product ion spectra with excitation at q = 0.236. In the former mixture the individual components were isolated with near 100% efficiency while in the latter mixture the isolation efficiency dropped to near 50% for the oxycodone component and to 80% for the other components. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparison of LID versus CID activation modes in tandem mass spectrometry of peptides

We report our contribution to the systematic investigation of peptide fragmentations performed on high‐performance Tof equipment, operating in MS and MS/MS modes, such as ESI‐QqTof and MALDI‐Tof/Tof instruments that are commonly available today in proteomic laboratories. Whereas the former analyzer's configuration provides low‐energy collision‐induced dissociations (CID), the latter allows tunable activation methods of the selected parent ion to induce either metastable laser‐induced dissociations (LID) or high‐energy CID (‘gas on spectra LID’). Fragmentation of the monoprotonated ion of 53 peptides (FW 807–2853 g/mol) was undertaken upon low‐energy CID on an ESI‐QTof mass spectrometer (Waters) as well as high‐energy CID and LID conditions on a MALDI Ultraflex mass spectrometer (Bruker). Systematic comparison of MS/MS spectra provided useful information on the performance of each piece of equipment for efficient peptide sequencing and also insights into the observed fragmentation behaviors. Copyright © 2008 John Wiley & Sons, Ltd.     

Multidimensional mass spectrometry to characterize degradation products generated during MALDI of polystyrenes prepared by controlled radical polymerization techniques

An analytical workflow involving high resolution mass analysis, collision‐induced dissociation and ion mobility was implemented to structurally characterize polymeric by‐products detected in lieu of intact species when performing matrix‐assisted laser desorption/ionization (MALDI) of polystyrenes with fragile end groups. Studied samples were prepared by atom transfer radical polymerization, reversible addition–fragmentation transfer polymerization and nitroxide‐mediated polymerization. Spectral resolution enabled by orthogonal injection of MALDI ions into a reflectron time‐of‐flight mass analyzer allowed a thorough inventory of species, including some with the same nominal m/z value but different elemental composition. Individual end‐group mass determination was achieved in MS/MS experiments, implementing an additional separative dimension based on ion mobility prior to CID to assist precursor ion selection in case of interferences. Besides validating commonly reported polystyrene chains terminated with either endo‐ or exo‐double bond, this multidimensional approach permitted to show that initiating moiety could also be affected by the MALDI process. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 3388–3397     

Differentiation of the Stereochemistry and Anomeric Configuration for 1-3 Linked Disaccharides Via Tandem Mass Spectrometry and <Superscript>18</Superscript>O-labeling

Collision-induced dissociation (CID) of deprotonated hexose-containing disaccharides (m/z 341) with 1–2, 1–4, and 1–6 linkages yields product ions at m/z 221, which have been identified as glycosyl-glycolaldehyde anions. From disaccharides with these linkages, CID of m/z 221 ions produces distinct fragmentation patterns that enable the stereochemistries and anomeric configurations of the non-reducing sugar units to be determined. However, only trace quantities of m/z 221 ions can be generated for 1–3 linkages in Paul or linear ion traps, preventing further CID analysis. Here we demonstrate that high intensities of m/z 221 ions can be built up in the linear ion trap (Q3) from beam-type CID of a series of 1–3 linked disaccharides conducted on a triple quadrupole/linear ion trap mass spectrometer. ¹⁸O-labeling at the carbonyl position of the reducing sugar allowed mass-discrimination of the “sidedness” of dissociation events to either side of the glycosidic linkage. Under relatively low energy beam-type CID and ion trap CID, an m/z 223 product ion containing ¹⁸O predominated. It was a structural isomer that fragmented quite differently than the glycosyl-glycolaldehydes and did not provide structural information about the non-reducing sugar. Under higher collision energy beam-type CID conditions, the formation of m/z 221 ions, which have the glycosyl-glycolaldehyde structures, were favored. Characteristic fragmentation patterns were observed for each m/z 221 ion from higher energy beam-type CID of 1–3 linked disaccharides and the stereochemistry of the non-reducing sugar, together with the anomeric configuration, were successfully identified both with and without ¹⁸O-labeling of the reducing sugar carbonyl group.     

Identification of Proteins and Phosphoproteins Using Pulsed Q Collision Induced Dissociation (PQD)

Pulsed Q collision induced dissociation (PQD) was developed to facilitate detection of low-mass reporter ions from labeling reagents (e.g., iTRΑQ) in peptide quantification using an LTQ mass spectrometer (MS). Despite the large number of linear ion traps worldwide, the use and optimization of PQD for protein identification have been limited, in part due to less effective ion fragmentation relative to the collision induced dissociation (CID). PQD expands the m/z coverage of fragment ions to the lower m/z range by circumventing the typical low mass cut-off of an ion trap MS. Since database searching relies on the matching between theoretical and observed spectra, it is not clear how ion intensity and peak number might affect the outcomes of a database search. In this report, we systematically evaluated the attributes of PQD mass spectra, performed intensity optimization, and assessed the benefits of using PQD on the identification of peptides and phosphopeptides from an LTQ. Based on head-to-head comparisons between CID (higher intensity) and PQD (better m/z coverage), peptides identified using PQD generally have Xcorr scores lower than those using CID. Such score differences were considerably diminished by the use of 0.1% m-nitrobenzyl alcohol (m-NBA) in mobile phases. The ion intensities of both CID and PQD were adversely affected by increasing m/z of the precursor, with PQD more sensitive than CID. In addition to negating the 1/3 rule, PQD enhances direct bond cleavage and generates patterns of fragment ions different from those of CID, particularly for peptides with a labile functional group (e.g., phosphopeptides). The higher energy fragmentation pathway of PQD on peptide fragmentation was further compared to those of CID and the quadrupole-type activation in parallel experiments.     

Relationship between Hammett's parameters and in silico density functional with tandem mass ESI‐CID fragmentation: Dihydropyridines as prototypes

Over the years, with the instrumental analysis evolution, the relationships between the carried‐out results with the data of theoretical analysis in silico and the Hammett's parameters have been reported. They have been very useful for chemical characterization of small organic molecules. Thus, this work aims at showing the feasibility and limitations for Hammett's and density functional theory applications in electrospray ionization–collision‐induced dissociation (ESI‐CID) fragmentation provision. For this, 13 dihydropyrimidinones para, meta, and orto monosubstituted were studied using ESI and CID in positive mode. As a result, it was observed that the main fragmentation includes the isocyanate and ethanol loses at low energy. Nevertheless, at higher energies, radical ions formed by McLafferty rearrangement were observed. The Hammett plots were correlated fragmentation profiles, showing good linearity for the [M + H]⁺, which does not occur to radical ions and carbocation's. These tendencies had demonstrated that the stability of protonate and activation energy of secondary ions changes with the pKa. The density functional theory studies indicated that, both nitrogen atoms in the dihydropyrimidinone's prototypes are capable of being protonated. However, the activation energy of fragmentation products is not changed. Therefore, this work has shown information, which can be useful to understand tandem mass spectrometry in ESI‐CID conditions for small organic molecules series. This is the first step for normalization of fragmentation pathway.     

Differentiation of lisinopril and its RSS diastereomer by liquid chromatography combined with collision‐induced dissociation mass spectrometry

A simple and sensitive liquid chromatography tandem multiple‐stage mass spectrometry (HPLC/MS/MS) method suitable for bulk lisinopril analysis was developed, by which lisinopril and its RSS isomer were separated and differentiated. In the collision‐induced dissociation (CID) mass spectra of the [M + H]⁺ ions, the abundance of the fragment ion of m/z 246 for lisinopril was about two times higher than the ion of m/z 245; however, the former fragment ion was noted to be a little lower than the latter for RSS isomer at all collision energies. In the CID mass spectra of the [M + Li]⁺ ion, the abundance of the rearrangement ion of m/z 315 for the RSS isomer was about three times higher than that for lisinopril. Furthermore, the difference was supported by the results of energy‐resolved mass spectrometry (ERMS) in the test range of collision energies. Similar differences were also observed between the CID mass spectra of lisinopril and RSS isomer methylester, which indicated that the RSS isomer could be rapidly characterized by the CID mass spectra of both the protonated and lithium adduct ion. Elemental compositions of all the ions were confirmed by Fourier Transform ion cyclotron resonance ESI mass spectrometry (FT‐ICR‐ESI/MS). In addition, theoretical computations were carried out to support the experimental results. Copyright © 2009 John Wiley & Sons, Ltd.     

Ion trap mass analysis at high pressure: an experimental characterization

In recent years, it has become increasingly interesting to understand the performance of mass spectrometers at pressures much higher than those employed with conventional operating conditions. This interest has been driven by several influences, including demand for the development of reduced‐power miniature mass spectrometers, desire for improved ion transfer into and through mass spectrometers, enhanced‐yield preparative mass separations, and mass filtering at the atmospheric pressure interface. In this study, an instrument was configured to allow for the performance characterization of a rectilinear ion trap (RIT) at pressures up to 50 mtorr with air used as the buffer gas. The mass analysis efficiency, mass resolution, isolation efficiency, and collision‐induced dissociation (CID) efficiency were evaluated at pressures ranging from 1 to 50 mtorr. The extent of degradation of mass resolution, isolation efficiency and ion stability as functions of pressure were characterized. Also, the optimal resonance ejection conditions were obtained at various pressures. Operations at 50 mtorr demonstrated improved CID efficiency in addition to peak widths of 2 and 5 m/z units (full width at half‐maximum, FWHM) for protonated caffeine (m/z 195) and Ultramark (m/z 1521) respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

MS/MS in structural analysis of an oviposition-deterring pheromone

A recently characterized oviposition-deterring pheromone (ODP, structure 1) of the European cherry fruit fly was used as a test case for probing the potential of tandem mass spectrometry (MS/MS) in structure elucidation as a stand-alone technique. The glycolipid-taurinate 1 was subjected to MS/MS analyses under a variety of conditions with and without preceding chemical degradation. Acidic methanolysis of 1 and subsequent in-batch derivatization (trideuterioacetylation) yielded methyl 2,3,4,6-tetrakis-O-trideuterioacetyl-glucopyranoside (2), methyl 8,15-bis-trideuterioacetoxy-palmitate (3), and taurine (4) as suitable target compounds for direct mixture analysis.Low energy collision induced dissociation (CID) on selected precursor ions (MS/MS on [M + H – CH₃OH]⁺ and [M + H]⁺ produced by fast atom bombardment (FAB)) allowed direct identification of 2 and 4, respectively, by comparison with appropriate reference ions. In the case of 3, low energy CID (desorption chemical ionization (DCI) instead of FAB, MS/MS on [M + H]⁺) permitted deduction of gross molecular structure, but failed to provide positional detail. In sharp contrast,high energy CID of trideuterioacetylated intact 1 (FAB-MS/MS on [M – H]^– ions of la) clearly revealed a linear 8,15-hydroxylated palmitic acid backbone. Less certain was assignment of 15-O-glucosylation by this approach.     

A comparison of the peptide fragmentation obtained from a reflector matrix-assisted laser desorption-ionization time-of-flight and a tandem four sector mass spectrometer

The types, extent, and overall distribution of peptide fragmentation produced by matrix-assisted laser desorption-ionization-postsource decay (MALDI-PSD) on a reflector time-of-flight mass spectrometer were compared with those obtained from high and low energy collision-induced dissociation (CID) on a four-sector mass spectrometer and from liquid secondary ion mass spectrometry (LSIMS) ion source fragmentation and LSIMS metastable ion (MI) decomposition on a two-sector mass spectrometer. The model peptides studied had sequences and compositions that yielded predominantly either N- or C-terminal fragmentation from CID. For des-Arg¹ and des-Arg⁹ bradykinin (i.e., H-PPGFSPFR-OH and H-RP-PGFSPF-OH, respectively), the types of fragment ions and the extent to which each type is formed in both MALDI-PSD and low energy CID spectra are remarkably similar. This observation suggests that both methods deposit comparable internal energies (IE) into [M + H]⁺ precursor ions. The distribution of N-terminal, C-terminal, immonium, and internal fragmentation from MALDI-PSD spectra of des-Arg¹ and des-Arg⁹ bradykinin did not change dramatically with respect to the terminal arginine position, contrary to those from LSIMS MI decomposition, high and low energy CID spectra. This observation in combination with the prominent immonium, internal, and minus 17 fragment ion types in PSD indicates that the imparted IE from MALDI and the 14 µs of flight time may promote steady-state decomposition kinetics. Fragmentation distributions of MALDI-PSD spectra are also similar to those in LSIMS spectra. This implies that the distribution of protonation sites in [M + H]⁺ is comparable for both techniques.     

Application of low energy CID in the determination of structures of [M – halogen]+ ions obtained from diethyl halosuccinates under electron impact

Low energy collision induced dissociation (CID) spectra indicate that m/z 173 ions formed by the loss of a halogen atom from diethyl chloro- and bromo-succinate under electron impact are mixtures of O-protonated diethyl maleate (>90%) and fumarate (<10 %). Hydrogen migration precedes the C-halogen bond cleavage in these cases. The low energy CID spectrum of the m/z 173 [M? I]⁺ ion obtained from diethyl iodosuccinate shows that only a small fraction of the [M? I]⁺ ions are the O-protonated species formed by hydrogen migration. The results of this study demonstrate the potential of low energy CID in the determination of structure (including configuration) of gas phase ions.     

Implementation of Dipolar Resonant Excitation for Collision Induced Dissociation with Ion Mobility/Time-of-Flight MS

An ion mobility/time-of-flight mass spectrometer (IMS/TOF MS) platform that allows for resonant excitation collision induced dissociation (CID) is presented. Highly efficient, mass-resolved fragmentation without additional excitation of product ions was accomplished and over-fragmentation common in beam-type CID experiments was alleviated. A quadrupole ion guide was modified to apply a dipolar AC signal across a pair of rods for resonant excitation. The method was characterized with singly protonated methionine enkephalin and triply protonated peptide angiotensin I, yielding maximum CID efficiencies of 44 % and 84 %, respectively. The Mathieu q_x,y parameter was set at 0.707 for these experiments to maximize pseudopotential well depths and CID efficiencies. Resonant excitation CID was compared with beam-type CID for the peptide mixture. The ability to apply resonant waveforms in mobility-resolved windows is demonstrated with a peptide mixture yielding fragmentation over a range of mass-to-charge (m/z) ratios within a single IMS-MS analysis.

Space-charge effects with mass-selective axial ejection from a linear quadrupole ion trap

Methods to reduce mass shifts caused by space charge with mass‐selective axial ejection from a linear quadrupole ion trap are investigated. For axial ejection, dipole excitation is applied to excite ions at q ≈ 0.85. The trapping radiofrequency (rf) voltage is scanned to bring ions of different m/z values into resonance for excitation. In the fringing field at the quadrupole exit, excited ions gain axial kinetic energy, overcoming the trapping potential, and are ejected from the trap. Space charge causes the frequencies of ion oscillation to decrease. Thus, greater rf voltages are required to bring ions into resonance for excitation and ejection, and the ions shift to higher apparent masses in a mass spectrum. At the same time, the peaks broaden, lowering resolution. The effects of injection q value, ejection q value, excitation amplitude, quadrupole dc voltages applied to the electrodes, applying an rf voltage to the exit lens, and scan speed, on mass shifts have been studied experimentally. Most experiments were done with only ions of protonated reserpine (m/z 609.3 and its isotopic peaks) in the trap. Some experiments were done with ions of protonated reserpine and ions of m/z 622 in the trap. In general, the mass shifts are reduced with higher ejection q values, higher excitation amplitudes, with quadrupole dc applied, and at higher scan speeds. The application of quadrupole dc appears to increase the ion cloud temperature, which lowers mass shifts. Thus, a proper choice of operating conditions can reduce, but not eliminate, mass shifts caused by space charge. Copyright © 2011 John Wiley & Sons, Ltd.     

Comprehensive characterization of Annonaceous acetogenins within a complex extract by HPLC‐ESI‐LTQ‐Orbitrap® using post‐column lithium infusion

Annonaceous acetogenins (AAGs) are a homogenous class of polyketides proposed as environmental neurotoxins. Previous dereplication studies of AAGs were limited by the use of low‐resolution mass spectrometers. Only poor information in terms of structures was provided due to the limited fragmentation of protonated or sodium cationized species. An innovative approach, using reversed‐phase high‐performance liquid chromatography coupled to a hybrid linear ion trap / orbitrap mass spectrometer (LTQ‐Orbitrap®), was therefore performed. Sensitivity was enhanced by post‐column infusion of lithium, since AAGs have a high affinity for this cation. High level of structural information was obtained from low‐energy‐collision‐induced dissociation fragmentation experiments of lithium‐cationized AAGs ([M + Li]⁺ ions) as demonstrated with purified standards. The method was then applied to a total ethyl‐acetate extract prepared from commercial soursop nectar (Annona muricata L.). The sensitivity, mass accuracy and specific fragmentation patterns proved to be particularly useful for characterization of the AAGs. Typical structural identification procedure and unexpected observations for specific structural types are illustrated, with major and minor compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

High‐throughput approaches towards the definitive identification of pharmaceutical drug metabolites. 2. An example of how unexpected dissociation behaviour could preclude correct assignment of sites of metabolism

S‐oxidation is a common metabolic route for sulfur‐containing compounds. Whilst investigating the dissociation of a series of chemically synthesised model S‐oxide metabolites, two unexpected losses of 62 m/z units were observed in the collision‐induced dissociation (CID) product ion spectrum of protonated 3‐dimethylaminomethyl‐4‐(4‐methanesulfinyl‐3‐methylphenoxy)benzenesulfonamide. A single loss was initially assigned using the low‐resolution product ion spectrum, acquired by electrospray ionisation quadrupole ion trap mass spectrometry (ESI‐QIT‐MS), as methanethial, S‐oxide via a charge‐remote, four‐centred rearrangement. This assignment was consistent with well‐documented hydrogen rearrangements in the literature. Further, the loss was not observed for the parent compound. Thus, it was inferred that the site of metabolism was involved in the dissociation and the attractive nature of the four‐centred rearrangement meant that the loss of methanethial, S‐oxide was a logical assignment. However, deuterium‐labelling experiments and accurate mass measurements, performed using electrospray ionisation Fourier transform ion cyclotron resonance mass spectrometry (ESI‐FT‐ICR‐MS), showed that the nominal loss of 62 m/z units occurs via two distinct dissociation pathways. Neither of these losses was of methanethial, S‐oxide as initially hypothesised from the low‐resolution product ion spectrum of the protonated molecule. Mechanisms consistent with the experimental findings are postulated. An MS³ spectrum of the fully exchanged, deuterated species supported the proposed mechanisms by suggesting that 3‐dimethylaminomethyl‐4‐(4‐methanesulfinyl‐3‐methylphenoxy)benzenesulfonamide has multiple sites of protonation in the gas phase. The planar structures of the posited product ions are likely to provide the driving force for the rearrangements. The relevance of the observations with regards to pharmaceutical drug metabolite identification is discussed. Copyright © 2009 John Wiley & Sons, Ltd.