Collision-induced Dissociation Research Articles

Mass Spectrometry Rearrangement Ions and Metabolic Pathway-Based Discovery of Indole Derivatives during the Aging Process in Citrus reticulata 'Chachi'.

The rapid analysis and characterization of compounds using mass spectrometry (MS) may overlook trace compounds. Although targeted analysis methods can significantly improve detection sensitivity, it is hard to discover novel scaffold compounds in the trace. This study developed a strategy for discovering trace compounds in the aging process of traditional Chinese medicine based on MS fragmentation and known metabolic pathways. Specifically, we found that the characteristic component of C. reticulata 'Chachi', methyl N-methyl anthranilate (MMA), fragmented in electrospray ionization coupled with collision-induced dissociation (CID) to produce the rearrangement ion 3-hydroxyindole, which was proven to exist in trace amounts in C. reticulata 'Chachi' based on comparison with the reference substance using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Combining the known metabolic pathways of 3-hydroxyindole and the possible methylation reactions that may occur during aging, a total of 10 possible indole derivatives were untargeted predicted. These compounds were confirmed to originate from MMA using purchased or synthesized reference substances, all of which were detected in C. reticulata 'Chachi' through LC-MS/MS, achieving trace compound analysis from untargeted to targeted. These results may contribute to explaining the aging mechanism of C. reticulata 'Chachi', and the strategy of using the CID-induced special rearrangement ion-binding metabolic pathway has potential application value for discovering trace compounds.

Derivatization procedure of estradiol with a combination of MPDNP-F and 4-dimethylaminopyridine to generate product ion containing estradiol-skeleton for reliable determination of its serum/plasma concentrations by LC/ESI-MS/MS

The quantification of serum/plasma estradiol (E2) is useful for the diagnosis, pathological analysis, and monitoring of the therapeutic efficacy of estrogen-dependent diseases. In this study, an improved derivatization method using 1-(2,4-dinitro-5-fluorophenyl)-4,4-dimethylpiperazinium iodide (MPDNP-F) was developed and combined with liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) for the sensitive and specific quantification of the serum/plasma E2. In the new method, the reaction time was reduced to 15 min from 90 min (two-step reaction in the previous method) by the direct reaction of MPDNP-F with E2 at 60°C in the presence of 4-dimethylaminopyridine (DMAP). DMAP served as the organic catalyst and had a less negative effect on the LC/ESI-MS/MS instrument compared to the non-volatile inorganic salt (NaHCO3), which was used in the previous method. The collision-induced dissociation of the molecular cation ([M]+) of the resulting derivative provided a product ion containing the E2-skeleton ([M–NO2–H]+), which significantly enhanced the assay sensitivity and specificity; compared to the dansyl chloride derivatization, which is the currently most-used derivatization procedure for the LC/ESI-MS/MS assays of E2, the MPDNP-F derivatization had significantly fewer interfering peaks and a clear and flat baseline in the serum sample analysis. The MPDNP-F derivatization–LC/ESI-MS/MS method enabled the precise and accurate quantification of E2 even at a 5.0 pg/mL concentration (lower limit of quantification) with a small sample volume (100 μL of serum/plasma) and had a tolerance for the matrix effect. This method was also proven to serve as a more sensitive and specific alternative to the clinically used chemiluminescence enzyme immunoassay.Graphical

A chemical derivatization-based pseudotargeted liquid chromatography-tandem mass spectrometry method for sensitive and high coverage determination of bile acids in human serum

Global profiling of bile acids (BAs) is imperative for understand their function and disease pathogenesis. But it is still a challenging task, as the collision-induced dissociation (CID) fragment ions of unconjugated BAs showed low ion intensities to insufficient analysis. Herein, we developed a highly sensitive method for pseudotargeted profiling of BAs by chemical derivatization. In the developed method, a labeling reagent, 2-dimethylaminoethylamine (DMED), was adopted to label the carboxyl group of BAs. The results demonstrated that the detection sensitivities of unconjugated BAs were increased by 4–200 folds after DMED-labeling. Moreover, to profile other potential BAs not included in the 91 known targets, diverse survey experiments were performed on Qtrap-MS to search BAs for both precursor and fragment ion species, and retention index (RI) strategy was adopted to facilitate the identification of isomers. Finally, MRM-based LC-MS/MS method was validated for the pseudotargeted profiling of the BAs submetabolome with good linearity (r2 ≥ 0.990 for 89 known BAs) and high sensitivity (0.05–0.5 ng/mL for unconjugated BAs), covering unconjugated, glycine, taurine, sulfuric acid, glucuronic acid, and as well as those doubly-conjugated with above types. With this method, a total of 107 BAs, covering 54 BAs identified by authentic standards and 53 BAs candidates, were successfully determined in human serum of women with intrahepatic cholestasis of pregnancy (ICP). Multivariate analysis revealed deferentially expressed BAs. ICP disease altered the BAs profile with a reduced proportion of unconjugated, sulfate- and doubly-conjugated BAs and an increased proportion of glycine and taurine conjugates. Altered proportion and profile of BAs in ICP groups were gradually recovered during the ursodeoxycholic acid (UDCA) therapy. Overall, the strategy of DMED-labeling technique combined with diverse survey experiments is sufficiently sensitive and robust to comprehensively analysis of metabolic profiling of BAs in human serum.

Structural Elucidation and Relative Quantification of Fatty Acid Double Bond Positional Isomers in Biological Tissues Enabled by Gas-Phase Charge Inversion Ion/Ion Reactions.

Fatty acids (FAs) contain a vast amount of structural diversity, and differences in fatty acid structure have been associated with various disease states. Accurate identification and characterization of fatty acids is critical to fully understand the biochemical roles these compounds play in disease progression. Conventional tandem mass spectrometry (MS/MS) workflows do not provide sufficient structural information, necessitating alternative dissociation methods. Gas-phase charge inversion ion/ion reactions can be used to alter the ion type subjected to activation to provide improved or complementary structural information. Herein, we have used an ion/ion reaction between fatty acid (FA) anions and magnesium tris-phenanthroline [Mg(Phen)3] dications to promote charge remote fragmentation of carbon-carbon bonds along the fatty acid chain, allowing for localization of carbon-carbon double bond (C=C) positions to successfully differentiate monounsaturated fatty acid isomers. Relative quantification was also performed to obtain the relative abundance of fatty acid isomers in different biological tissues. For example, the relative abundance of FA 18:1 (9) was determined to vary across regions of rat brain, rat kidney, and mouse pancreas, and FA 16:1 (9) was found to have a higher relative abundance in the dermis layer compared to the sebaceous glands in human skin tissue.

Properties of Metal Hydrides of the Iron Triad.

Metal hydride complexes are essential intermediates in hydrogenation reactions. The hydride-donor ability determines the scope of use of these complexes. We present a new, simple mass-spectrometry method to study the hydride-donor ability of metal hydrides using a series of 18 iron, cobalt, and nickel complexes with N- and P-based ligands (L). The mixing of [(L)MII(OTf)2] with NaBH4 forms [(L)MII(BH4)]+ (M = Fe, Co, Ni) that can be detected by electrospray ionization mass spectrometry. Energy-resolved collision-induced dissociations of [(L)MII(BH4)]+ provide threshold energies (ΔECID) for the formations of [(L)MII(H)]+ that correlate well with the hydride donor ability of the metal hydride complexes. We studied the vibrational and electronic spectra of the generated metal hydrides, assigned their structure and spin state, and demonstrated a good correlation between ΔECID and the M-H stretching vibration frequencies. The ΔECID also correlates with reaction rates for hydride transfer reactivity in the gas phase and known reactivity trends in the solution phase.

Unraveling the Unimolecular Ion Chemistry of Protonated Isoprene and Prenol.

The atmospheric chemistries of isoprene and prenol have been studied extensively; however, much of that research has focused on neutral or radical chemistry. Recent studies have demonstrated that under acidic conditions, isoprene and prenol can become protonated in the atmosphere, and we have explored the unimolecular chemistry of protonated isoprene and prenol with tandem mass spectrometry (using a triple-quadrupole mass spectrometer) and density functional theory. The collision-induced dissociation of protonated isoprene revealed two product ion channels: the neutral losses of C2H4 and H2, the former dominating over the latter. Protonated prenol dissociates by four product ion channels: the neutral losses of water, formaldehyde, methanol, and propene, with the former two being minor channels and the latter two being major channels. Density functional theory supplemented with CBS-QB3 single-point calculations revealed the underlying mechanisms to explain the breakdown behavior. The two competing channels from protonated isoprene could easily be rationalized due to the relative energy difference between key transition states along the reaction coordinates. However, in the case of protonated prenol, it was revealed that the minor products observed in the breakdown of protonated prenol had significantly lower reaction barriers when compared to the major products, an apparent contradiction. This could be rationalized if the initial ion population entering the collision cell is comproed of several isomeric species on the minimum energy reaction pathway, species populated by collisional excitation in the ion source region.

Unraveling the cytotoxicity and cellular uptake of low, medium and high molecular weight polyethylene glycol polymers in MCF-7 cells by green UPLC-MS/MS methods

Unraveling the cytotoxicity and cellular uptake of low, medium and high molecular weight polyethylene glycol (PEG) in cells is important for evaluation of therapeutic efficacy and toxicity of PEGylated drug delivery systems. In this study, cellular uptake of PEG600, PEG2K, PEG4K and PEG10K in MCF-7 cells was first studied by an UPLC-MS/MS assay coupled with collision induced dissociation (CID) in source technique. The CID of PEG in source with high values of declustering potentials generates numerous PEG-related product ions. These PEG-related fragment ions can be further broken into specific product ions in the collision cell as alternative ions for detection of PEG. The quantification of PEG was finally performed with the MRM transition (m/z 221.0 → 89.0). The experimental results indicated that the toxicity of PEG600, PEG2K, PEG4K and PEG10K was not significant at concentrations of 5–1200 μg/mL and the amounts of PEG polymers entry into MCF-7 cells at was small. The greenness of the developed analytical methods was also assessed by Analytical Eco-Scale, Analytical Greenness calculator (AGREE) and Green Analytical Procedure Index (GAPI) in this study.

Computational Expansion of High-Resolution-MSn Spectral Libraries.

Commonly, in MS-based untargeted metabolomics, some metabolites cannot be confidently identified due to ambiguities in resolving isobars and structurally similar species. To address this, analytical techniques beyond traditional MS2 analysis, such as MSn fragmentation, can be applied to probe metabolites for additional structural information. In MSn fragmentation, recursive cycles of activation are applied to fragment ions originating from the same precursor ion detected on an MS1 spectrum. This resonant-type collision-activated dissociation (CAD) can yield information that cannot be ascertained from MS2 spectra alone, which helps improve the performance of metabolite identification workflows. However, most approaches for metabolite identification require mass-to-charge (m/z) values measured with high resolution, as this enables the determination of accurate mass values. Unfortunately, high-resolution-MSn spectra are relatively rare in spectral libraries. Here, we describe a computational approach to generate a database of high-resolution-MSn spectra by converting existing low-resolution-MSn spectra using complementary high-resolution-MS2 spectra generated by beam-type CAD. Using this method, we have generated a database, derived from the NIST20 MS/MS database, of MSn spectral trees representing 9637 compounds and 19386 precursor ions where at least 90% of signal intensity was converted from low-to-high resolution.

Isolation and Identification of 12-Deoxyphorbol Esters from Euphorbia resinifera Berg Latex: Targeted and Biased Non-Targeted Identification of 12-Deoxyphorbol Esters by UHPLC-HRMSE.

Diterpenes from the Euphorbia genus are known for their ability to regulate the protein kinase C (PKC) family, which mediates their ability to promote the proliferation of neural precursor cells (NPCs) or neuroblast differentiation into neurons. In this work, we describe the isolation from E. resinifera Berg latex of fifteen 12-deoxyphorbol esters (1-15). A triester of 12-deoxy-16-hydroxyphorbol (4) and a 12-deoxyphorbol 13,20-diester (13) are described here for the first time. Additionally, detailed structural elucidation is provided for compounds 3, 5, 6, 14 and 15. The absolute configuration for compounds 3, 4, 6, 13, 14 and 15 was established by the comparison of their theoretical and experimental electronic circular dichroism (ECD) spectra. Access to the above-described collection of 12-deoxyphorbol derivatives, with several substitution patterns and attached acyl moieties, allowed for the study of their fragmentation patterns in the collision-induced dissociation of multiple ions, without precursor ion isolation mass spectra experiments (HRMSE), which, in turn, revealed a correlation between specific substitution patterns and the fragmentation pathways in their HRMSE spectra. In turn, this allowed for a targeted UHPLC-HRMSE analysis and a biased non-targeted UHPLC-HRMSE analysis of 12-deoxyphorbols in E. resinifera latex which yielded the detection and identification of four additional 12-deoxyphorbols not previously isolated in the initial column fractionation work. One of them, identified as 12-deoxy-16-hydroxyphorbol 20-acetate 13-phenylacetate 16-propionate (20), has not been described before.

Development of stability-indicating HPLC method for quantification of pharmacopeia impurities of Zuclopenthixol and characterization of its stress degradation products by LCMS/MS

BackgroundThe present study focused to develop a simple and sensitive HPLC method for resolution and estimation process-related impurities of zuclopenthixol and further assessment of forced degradation behavior of zuclopenthixol.ResultsThe chromatographic separation of drug substance, process-related impurities and its degradation products (DPs) was achieved on KNAUER C18 (250 mm × 4.6 mm, 5µ id) column at that was maintained at 35 °C temperature using 0.1 M sodium acetate buffer at pH 4.3 and methanol in 20:80 (v/v) as mobile phase A, 0.1% formic acid and acetonitrile in 75:25 (v/v) as mobile phase B. Equal volume of mobile phase A and B was pumped in isocratic elution at 0.8 mL/min. Detection wavelength was selected as 257 nm. In the proposed conditions, zuclopenthixol is identified at 6.91 and 1.91 min and 2.89 min, respectively, for impurity B and A min with acceptable system suitability and specificity. The method produces LOD at 0.009 for impurities with calibration range of 30–180 µg/mL for zuclopenthixol and 0.03–0.18 µg/mL for impurities. The other validation parameters were notices to be with in the acceptable levels for zuclopenthixol and its impurities. The drug was exposed to different stressed conditions (acid, base, peroxide, thermal and UV light) according to ICH Q1A (R2) guidelines. The DPs formed during the stress study were identified and characterized by LCMS/MS in ESI positive mode.ConclusionThe analysis involved a comparison of collision-induced dissociation mass spectrometry data between the degradation products and zuclopenthixol. As a result, potential structures for six degradation compounds were suggested. The results from additional validation studies were similarly pleasing and demonstrated their suitability for the routine analysis of zuclopenthixol and its associated impurities in both bulk drug and pharmaceutical dosage forms. Additionally, these findings can be extended to assess the mechanism of stress degradation in zuclopenthixol.

Development of an Automated, High-Throughput Methodology for Native Mass Spectrometry and Collision-Induced Unfolding.

Native ion mobility mass spectrometry (nIM-MS) has emerged as a useful technology for the rapid evaluation of biomolecular structures. When combined with collisional activation in a collision-induced unfolding (CIU) experiment, nIM-MS experimentation can be leveraged to gain greater insight into biomolecular conformation and stability. However, nIM-MS and CIU remain throughput limited due to nonautomated sample preparation and introduction. Here, we explore the use of a RapidFire robotic sample handling system to develop an automated, high-throughput methodology for nMS and CIU. We describe native RapidFire-MS (nRapidFire-MS) capable of performing online desalting and sample introduction in as little as 10 s per sample. When combined with CIU, our nRapidFire-MS approach can be used to collect CIU fingerprints in 30 s following desalting by using size exclusion chromatography cartridges. When compared to nMS and CIU data collected using standard approaches, ion signals recorded by nRapidFire-MS exhibit identical ion collision cross sections, indicating that the same conformational populations are tracked by the two approaches. Our data further suggest that nRapidFire-MS can be extended to study a variety of biomolecular classes, including proteins and protein complexes ranging from 5 to 300 kDa and oligonucleotides. Furthermore, nRapidFire-MS data acquired for biotherapeutics suggest that nRapidFire-MS has the potential to enable high-throughput nMS analyses of biopharmaceutical samples. We conclude by discussing the potential of nRapidFire-MS for enabling the development of future CIU assays capable of catalyzing breakthroughs in protein engineering, inhibitor discovery, and formulation development for biotherapeutics.

Encapsulation in Charged Droplets Generates Distorted Host-Guest Complexes.

The ability of various hydrogen-bonded resorcinarene-based capsules to bind α,ω-alkylbisDABCOnium (DnD) guests of different lengths was investigated in solution and in the gas-phase. While no host-guest interactions were detected in solution, encapsulation could be achieved in the charged droplets formed during electrospray ionisation (ESI). This included guests, which are far too long in their most stable conformation to fit inside the cavity of the capsules. A combination of three mass spectrometric techniques, namely, collision-induced dissociation, hydrogen/deuterium exchange, and ion-mobility mass spectrometry, together with computational modelling allow us to determine the binding mode of the DnD guests inside the cavity of the capsules. Significant distortions of the guest into horseshoe-like arrangements are required to optimise cation-π interactions with the host, which also adopt distorted geometries with partially open hydrogen-bonding seams when binding longer guests. Such quasi "spring-loaded" capsules can form in the charged droplets during the ESI process as there is no competition between guest encapsulation and ion pair formation with the counterions that preclude encapsulation in solution. The encapsulation complexes are sufficiently stable in the gas-phase - even when strained - because non-covalent interactions significantly strengthen in the absence of solvent.

Diagnostic utility of N-terminal TMPP labels for unambiguous identification of clipped sites in therapeutic proteins

Protein therapeutics are susceptible to clipping via enzymatic and nonenzymatic mechanisms that create neo-N-termini. Typically, neo-N-termini are identified by chemical derivatization of the N-terminal amine with (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) followed by proteolysis and mass spectrometric analysis. Detection of the TMPP-labeled peptide is achieved by mapping the peptide sequence to the product ion spectrum derived from collisional activation. The site-specific localization of the TMPP tag enables unambiguous determination of the true N-terminus or neo-N-termini. In addition to backbone product ions, TMPP reporter ions at m/z 573, formed via collision-induced dissociation, can be diagnostic for the presence of a processed N-termini. However, reporter ions generated by collision-induced dissociation may be uninformative because of their low abundance. We demonstrate a novel high-throughput LC–MS method for the facile generation of the TMPP reporter ion at m/z 533 and, in some instances m/z 590, upon electron transfer dissociation. We further demonstrate the diagnostic utility of TMPP labeled peptides derived from a total cell lysate shows high degree of specificity towards selective N-terminal labeling over labeling of lysine and tyrosine and highly-diagnostic Receiver Operating Characteristic’s (ROC) of TMPP reporter ions of m/z 533 and m/z 590. The abundant generation of these reporters enables subsequent MS/MS by intensity and m/z-dependent triggering of complementary ion activation modes such as collision-induced dissociation, high-energy collision dissociation, or ultraviolet photo dissociation for subsequent peptide sequencing.

Mass spectrometry-based gas phase intramolecular benzyl migration in sparsentan, a novel endothelin and angiotensin II receptor antagonist.

We report a collision-induced dissociation (CID) based gas phase rearrangement study using quadrupole time-of-flight mass spectrometry coupled with liquid chromatography on a novel endothelin and angiotensin II receptor antagonist, sparsentan. We performed tandem mass spectrometry to identify precursor and fragment ion relationships and assigned structures for major fragment ions. We propose a benzyl migration mechanism based on bond length measurements in density functional theory (B3LYP/6-31+G*) optimized geometries of protonated sparsentan and its m/z 547 fragment. Protonated sparsentan undergoes loss of ethanol, which yields a resonance-stabilized benzylic cation with m/z 547, which further fragments into m/z 353 via benzyl migration, where the benzylic cation migrates to one of the nucleophilic nitrogen atoms followed by proton transfer from the sulfonamide nitrogen to a carbonyl oxygen, resulting in a neutral loss of mass 194. Further fragmentation of m/z 353 results in m/z 258, which undergoes radical and neutral loss to yield m/z 193 and 194, respectively. The proposed mechanism of generation of m/z 353 was confirmed by CID of deuterated sparsentan. Considering the importance of gas phase rearrangements of organic molecules in structural identifications as well as the novelty of the molecule, these findings will be helpful for future studies to predict gas phase benzyl migration in sparsentan analogs and for degradation product and metabolite identification of sparsentan and its analogs using LC-MS.

Interactions of CyMe4 -BTBP ligand with lanthanides and actinides: Insights from ESI-MS and DFT calculations.

Electrospray Ionization Mass Spectrometry (ESI-MS) technique and density functional theory (DFT) calculations were combined to study the formation of the complexes of lanthanides (Ln = La, Ce, Nd, Sm, Eu, Yb) and actinides (UO2 2+ , Th4+ ) with CyMe4 -BTBP (6,6'-bis(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-benzo-[1,2,4-]triazin-3-yl)-[2,2']bipyridine) to understand the mechanisms during the extraction process. Mass spectrometry titrations showed the formation of the complexation in acetonitrile. For lanthanides, only 1:2 complexes ([Ln(L)2 ]3+ , [Ln(L)2 (CH3 CN)]3+ ), [Ln(L)2 (NO3 )]2+ ) were found at low [Ln]/[L] concentration ratios, whereas the 1:1 complexes ([Ln(L)(NO3 )2 ]+ ) were observed when the [Ln]/[L] concentration ratio reached 1.0. For uranyl complexes, 1:1 complex ([UO2 L(NO3 )]+ ) was the only species within the measuring range. Th4+ complexes had two compositions: 1:1 and 1:2, in which 1:2 species was the dominant complex. Collision-induced dissociation (CID) was employed to characterize the fragmentation process. The fragmentation process was unfolded sequentially on both sides of CyMe4 -BTBP ligand with the loss of alkyl groups and cleavage of triazinyl rings. The CID results of CyMe4 -BTBP complexes revealed a slight difference depending on the metal center. The DFT calculations showed that the stable complexes formed in acetonitrile solution were consistent with the ESI-MS results.

Radical-Induced Dissociation for Oligonucleotide Sequencing by TiO2/ZnAl-Layered Double Oxide-Assisted Laser Desorption/Ionization Mass Spectrometry.

De novo sequencing of oligonucleotides remains challenging, especially for oligonucleotides with post-transcriptional or synthetic modifications. Mass spectrometry (MS) sequencing can reliably detect and locate all of the modification sites in oligonucleotides via m/z variance. However, current MS-based sequencing methods exhibit complex spectra and low ion abundance and usually require coupled instrumentation. Herein, we demonstrate a method of oligonucleotide sequencing using TiO2/ZnAl-layered double oxide (LDO)-assisted laser desorption/ionization (LDI)-MS based on radical-induced dissociation (RID). ·CH2OH radicals can be produced on the surface of a TiO2/ZnAl-LDO matrix via ultraviolet light, inducing an attack on the active site of the oligonucleotide phosphate skeleton to create typical "a-, a-B-, c·-, d-, w-, and y"-type fragments. Compared with the spectra obtained via collision-based methods, such as collision-induced dissociation and higher-energy collisional dissociation, the LDI-MS spectra based on RID exhibit single-charged signals, fewer types of fragments, and a lower proportion of unknown noise peaks. We demonstrate full sequence coverage for a 6-mer 2'-O-methyl-modified oligonucleotide and a 21-mer small interfering RNA and show that RID can sequence oligonucleotides with modifications. Importantly, the mechanism responsible for the RID of the oligonucleotide phosphate skeleton was investigated through offline experiments, demonstrating consistent results with density functional theory calculations.

Convenient high resolution mass spectrometry characterization of aromatic sulfur-containing petroleum components following by preliminary S-alkylation with aliphatic alcohols

This paper describes rather suitable and variable preliminary derivatization strategy that may precede the molecular level characterization of sulfur-containing compounds of a particularly aromatic nature by high-resolution MALDI and ESI mass spectrometry. We demonstrated for the first time that free aliphatic alcohols (primary 1-alkanols C3–C20) in the presence of triflic acid provide easy S-alkylation of not only saturated sulfides but also most typical aromatic sulfur-containing compounds (benzothiophene, dibenzothiophene and their homologues) widely distributed and frequently analyzed in oil. The reaction proceeds quantitatively at rather mild conditions and gives rise to corresponding S-alkyl sulfonium salts the cation moieties of which can be detected using MALDI and ESI mass spectrometry with excellent signal/noise (S/N) ratios; the response ratios for target ions being quite close for both methods. Collision-induced dissociation (CID) of S-alkylsulfonium cations proceeds only by the elimination of entire S-alkyl group yielding protonated molecule of the analyte. This process can be useful for a reliable determination of target aromatic heterocyclic compounds in complex mixtures. The applicability of the method is illustrated by the analysis of deasphalted medium petroleum sample. The proposed derivatization principle is considered to be highly applicable as an alternative approach to routine characterization and sensitive determination of most typical sulfur-containing compounds and particularly of aromatic S-heterocycles in crude oils by soft-ionization mass spectrometry methods.

Electron activated dissociation - a complementary fragmentation technique to collision-induced dissociation for metabolite identification of synthetic cathinone positional isomers

Over the last decade, a remarkable number of new psychoactive substances (NPS) have emerged onto the drug market, resulting in serious threats to both public health and society. Despite their abundance and potential toxicity, there is little information available on their metabolism, a crucial piece of information for clinical and forensic purposes. NPS metabolism can be studied using in vitro models, such as liver microsomes, cytosol, hepatocytes, etc. The tentative structural elucidation of metabolites of NPS formed using in vitro models is typically carried out using liquid chromatography combined with high-resolution tandem mass spectrometry (LC-HRMS2) with collision-induced dissociation (CID) as a fragmentation method. However, the thermally-excited ions produced with CID may not be sufficient for unambiguous identification of metabolites or their complete characterization. Electron-activated dissociation (EAD), a relatively new fragmentation approach that can be used to fragment singly-charged ions, may provide complementary structural information that can be used to further improve the confidence in metabolite identification. The aim of this study was to compare CID and EAD as fragmentation methods for the characterization and identification of synthetic cathinone positional isomers and their metabolites. The in vitro metabolism of 2-methylethcathinone (2-MEC), 3-methylethcathinone (3-MEC) and 4-methylethcathinone (4-MEC) was investigated with both CID and EAD methods using LC-HRMS2. Four, seven and six metabolites were tentatively identified for the metabolism of 2-MEC, 3-MEC and 4-MEC, respectively. Here, the metabolism of 3-MEC and 2-MEC is reported for the first time. The EAD product ion mass spectra showed different fragmentation patterns compared to CID, where unique and abundant product ions were observed in EAD but not in CID. More importantly, certain EAD exclusive product ions play a significant role in structural elucidation of some metabolites. These results highlight the important role that EAD fragmentation can play in metabolite identification workflows, by providing additional fragmentation data compared with CID and, thus, enhancing the confidence in structural elucidation of drug metabolites.

Conformationally Restricted Glycopeptide Backbone Inhibits Gas-Phase H/D Scrambling between Glycan and Peptide Moieties.

Protein glycosylation is a common post-translational modification on extracellular proteins. The conformational dynamics of several glycoproteins have been characterized by hydrogen/deuterium exchange mass spectrometry (HDX-MS). However, it is, in most cases, not possible to extract information about glycan conformation and dynamics due to the general difficulty of separating the deuterium content of the glycan from that of the peptide (in particular, for O-linked glycans). Here, we investigate whether the fragmentation of protonated glycopeptides by collision-induced dissociation (CID) can be used to determine the solution-specific deuterium content of the glycan. Central to this concept is that glycopeptides can undergo a facile loss of glycans upon CID, thereby allowing for the determination of their masses. However, an essential prerequisite is that hydrogen and deuterium (H/D) scrambling can be kept in check. Therefore, we have measured the degree of scrambling upon glycosidic bond cleavage in glycopeptides that differ in the conformational flexibility of their backbone and glycosylation pattern. Our results show that complete scrambling precedes the glycosidic bond cleavage in normal glycopeptides derived from a glycoprotein; i.e., all labile hydrogens have undergone positional randomization prior to loss of the glycan. In contrast, the glycosidic bond cleavage occurs without any scrambling in the glycopeptide antibiotic vancomycin, reflecting that the glycan cannot interact with the peptide moiety due to a conformationally restricted backbone as revealed by molecular dynamics simulations. Scrambling is also inhibited, albeit to a lesser degree, in the conformationally restricted glycopeptides ristocetin and its pseudoaglycone, demonstrating that scrambling depends on an intricate interplay between the flexibility and proximity of the glycan and the peptide backbone.

Radial Electric Field Driven Collision-Induced Dissociation in a Miniature Continuous Atmospheric Pressure Interfaced Ion Trap Mass Spectrometer.

Miniature ion trap mass spectrometry with the unique ability of tandem-in-time analysis is extensively used in public security and environmental pollution detection. In this study, a novel radial electric field driven collision-induced dissociation (REFD-CID) is presented with high fragmentation efficiency for different species by adjusting the float DC, the initial kinetic energy of ions, and the pressure in a miniature continuous atmospheric pressure interfaced ion trap mass spectrometer (CAPI-ITMS). It is noteworthy that multiple fragment ions ([M+H-nC2H4]+, where n = 1, 2, 3) of triethyl phosphate were observed with a single injection of ions. The underlying mechanism of the REFD-CID revealed that the enhanced radial electric field by modulation of the float DC drove ions toward regions of intense RF field where broadband heating and dissociation of ions took place through theoretical simulations. Finally, the REFD-CID was utilized to improve the instrument performances. The existence of reagent ions led to a severe space charge effect as the ion injection duration of the CAPI-ITMS was extended to enhance the sensitivity of aniline. Through selective fragmentation of reagent ions, peak broadening and mass shift were eliminated, and meanwhile, a 28-fold improvement of aniline in a signal-to-noise ratio was achieved with the ion injection duration varying from 50 to 2500 ms. Moreover, isomeric illicit drugs (JWH-018 and acetylcodeine) were distinguished by generating multiple characteristic fragment ions, demonstrating potential applications in the identification of synthetic illicit drugs.