In-source Fragmentation Research Articles

Rapid screening of glycoalkaloids in Solanum scabrum and S. nigrum berries using ultra-high-performance liquid chromatography with pathway-specified in-source fragmentation tandem mass spectrometry.

The safe consumption of Solanum scabrum and S. nigrum berries (SNBs) depends on a reliable and rapid chemical screen for the testing of the fruit and/or final food and industrial products for the presence and level of toxic glycoalkaloids. Such a rapid and sensitive screen could also be used by those involved in food safety and forensics, industry, research labs and those in agriculture production, breeding and food processing. Significant variation in the content and composition of glycoalkaloids across SNBs has been reported. To facilitate high-throughput targeted analysis, this work overcame the slow scan speed of a traditional triple quadruple mass spectrometry (QqQ) method by development of a pseudo-MS3 method. In-source fragmentation functioned as a pseudo-MS or pseudo-hydrolysis to trim down the structurally diverse and complex glycosides into five types of aglycone ions, which were then analyzed using multiple reaction monitoring (MRM). Characteristic product ions were selected based on the aglycone skeleton and substitution pattern and associated fragmentation pathway. A compact method with only 15 MRM transitions were developed for high-throughput screening of very diverse glycoalkaloids. Glycosides of the same aglycone type were readily identified in the same transition window without the need for mass spectra interpretation. Validated using solamargine, the sole available standard, the accuracy was 99.7-101.3%, the intra- and inter-day precision were, respectively, 2.5-5.0% and 8.0-9.2%, and the lower limit of detection and quantification were, respectively, 3.1 and 10.2 ng/mL (with 1 μL injection volume). The peudo-MS3 method allowed for high-throughput targeted analysis with compact MRM transitions to address a large number of glycoalkaloids with diverse structures. This method could serve to meet the most heavy-duty demand for rapid inspection of glycoalkaloids in SNBs. This method can be adopted and used by those involved in food safety and forensics, in developing food and industrial products and in genetics and breeding.

In-Source Fragmentation of Phenethylamines by Electrospray Ionization Mass Spectrometry: Toward Highly Sensitive Quantitative Analysis of Monoamine Neurotransmitters.

Electrospray ionization mass spectrometry (ESI-MS) is widely used to analyze biomolecules, which are usually detected as protonated and cation-adducted molecules in the positive-ion mode. However, phenethylamine derivatives, which are known as neurotransmitters and psychoactive drugs, undergo the protonation and subsequently lose NH3 during ESI. As a result, intense fragment-ion signals are observed in their ESI-MS spectra, which hamper the unambiguous identification of phenethylamine derivatives. To understand the mechanism of the loss of NH3 from these phenethylammoniums, the fragmentations of model 4-substituted phenethylamines were investigated and the fragment ions were identified as spiro[2.5]octadienyliums. Fragmentation was enhanced by the presence of electron-donating groups, and most substituted phenethylamines generated spiro[2.5]octadienyliums as fragment ions during ESI-MS, except those with strong electron-withdrawing groups. The quantitative analysis of phenethylamines by liquid chromatography tandem mass spectrometry is typically performed by multiple reaction monitoring using protonated molecules as the precursor. In contrast, the conversion of precursor ions from the protonated molecules into the spiro[2.5]octadienylium fragment improved the signal-to-noise ratio, allowing the quantitative analysis of phenethylamines with high sensitivity and accuracy.

Ultrahigh-Resolution Mass Spectrometry Method for Resolving 13C-Enrichment Patterns in a Microalgal Lipidome.

The analysis of 13C-labeled lipids by mass spectrometry is challenging due to the complexity from labeling the large number of carbon atoms in lipids. To further add to the complexity, different adducts can be produced during electrospray ionization and in-source fragmentation, which can create complex overlapping isotope patterns that can only be resolved using high-resolution mass spectrometry. Co-elution of lipids even after chromatographic separation also adds to the potential for overlapping mass spectra. Here, we describe a procedure that enables full 13C-labeled patterns to be resolved in complex microalgal lipid extracts as well a procedure that provides structural labeling information. Mass resolving powers of 240000 full width half-maximum (fwhm) and fast targeted MS/MS allowed the differentiation of isotopologues, adducts, and unresolved lipid species after chromatographic separation. This enabled the percentage of 13C enrichment to be calculated for each individual lipid species over a time series in the microalgal lipidome. The application of tandem mass spectrometry (MS/MS) also allowed the degree of labeling within the headgroup vs acyl chains to be determined, further adding to the detail of information collected. This information is particularly useful for studying lipid synthesis and remodeling processes and can be extended to other biological systems.

Comparison of Heated Electrospray Ionization and Nanoelectrospray Ionization Sources Coupled to Ultra-High-Resolution Mass Spectrometry for Analysis of Highly Complex Atmospheric Aerosol Samples.

Direct infusion analysis using soft ionization techniques coupled to ultra-high-resolution mass spectrometers (UHRMS) allows screening of thousands of organic species in complex samples. Despite the high analytical throughput of direct infusion, this technique is known to be prone to matrix effects caused by changes in the ionization efficiency of an analyte, ion suppression, or enhancement due to the presence of certain compounds and inorganic salts in the sample. In this study we compared two soft ionization sources, that is, heated electrospray ionization (HESI) and nano-ESI for the analysis of atmospheric aerosol samples in the negative ionization mode. In-source fragmentation tests were conducted and experiments involving sample desalting through solid-phase extraction (SPE) with a reversed phase functionalized polymeric sorbent and spiking samples with inorganic salt were performed. Both ionization sources showed specific advantages and disadvantages for the direct infusion analysis of atmospheric aerosol extracts. The mass spectra of aerosol samples analyzed using HESI contained a large number of high molecular weight homologues containing sulfur and nitrogen, suggesting that this source is prone to formation of salt adducts and noncovalent compounds in samples enriched with inorganic salts. Data from the same aerosol sample extracts analyzed using nanoelectrospray ionization (nano-ESI) show less adduct formation; however, a decrease in the number of homologues was observed, as well as loss of molecules at higher mass range, indicating that the nano-ESI source is more prone to ion suppression. Irrespective of ionization source, SPE pretreatment significantly improved ion recoveries for organic species with nonpolar and moderately polar functional groups, but lower recoveries were obtained for highly oxygenated molecules. Therefore, while SPE reduced in-source adduct formation, it also limited the range of compounds identified through a single analysis.

Mass spectral reconstruction of LC/MS data with entropy minimization

Liquid chromatography-mass spectrometry (LC/MS) is a major technique for the analysis of complex samples. Data processing for LC/MS untargeted analysis is very challenging due to the presence of asymmetric, co-eluted at trace-level peaks. We report a multivariate curve resolution approach based on entropy minimization to resolve LC/MS data. Our liquid chromatography band-target entropy minimization (lc-BTEM) approach enables automated resolution of complex LC/MS peaks without the requirement of any parameter inputs. We demonstrated the performance of our approach on a co-eluted and trace-level peaks in an American ginseng extract. Consequently, we were able to putatively annotate the presence of more than 50 ginsenosides. The extracted pure mass spectra and in-source fragments information further enabled the annotation of [M+H]+, [M+H-Neu]+ and [M-H]- ions. This approach is expected to benefit LC/MS untargeted analysis and data independent analysis of complex samples.

Challenges and recommendations in developing LC-MS/MS bioanalytical assays of labile glucuronides and parent compounds in the presence of glucuronide metabolites.

Glucuronides, especially acyl glucuronides, were often found to be unstable in vitro and in vivo. Acyl glucuronide metabolites can convert back to the parent drugs at physiological pH through hydrolysis. Glucuronides can also undergo in-source fragmentation during MS ionization to form the same ions as those of the parent compounds, which could cause interference to the analysis of the parent compounds. All of these may cause significant challenges in developing LC-MS/MS bioanalytical assays of labile glucuronides or parent compounds in the presence of glucuronide metabolites. In this manuscript, we will discuss these challenges and summarize recommended strategies and practices for fast and efficient method development. Critical considerations in assay development will also be discussed.

Enhanced in-Source Fragmentation Annotation Enables Novel Data Independent Acquisition and Autonomous METLIN Molecular Identification.

Electrospray ionization (ESI) in-source fragmentation (ISF) has traditionally been minimized to promote precursor molecular ion formation, and therefore its value in molecular identification is underappreciated. In-source annotation algorithms have been shown to increase confidence in putative identifications by using ubiquitous in-source fragments. However, these in-source annotation algorithms are limited by ESI sources that are generally designed to minimize ISF. In this study, enhanced in-source fragmentation annotation (eISA) was created by tuning the ISF conditions to generate in-source fragmentation patterns comparable with higher energy fragments generated at higher collision energies as deposited in the METLIN MS/MS library, without compromising the intensity of precursor ions (median loss ≤10% in both positive and negative ionization modes). The analysis of 50 molecules was used to validate the approach in comparison to MS/MS spectra produced via data dependent acquisition (DDA) and data independent acquisition (DIA) mode with quadrupole time-of-flight mass spectrometry (QTOF-MS). Enhanced ISF as compared to QTOF DDA enabled higher peak intensities for the precursor ions (median: 18 times in negative mode and 210 times in positive mode), with the eISA fragmentation patterns consistent with METLIN for over 90% of the molecules with respect to fragment relative intensity and m/z. eISA also provides higher peak intensity as opposed to QTOF DIA for over 60% of the precursor ions in negative mode (median increase: 20%) and for 88% of the precursor ions in positive mode (median increase: 80%). Molecular identification with eISA was also successfully validated from the analysis of a metabolic extract from macrophages. An interesting side benefit of enhanced ISF is that it significantly improved molecular identification confidence with low resolution single quadrupole mass-spectrometry-based untargeted LC/MS experiments. Overall, enhanced ISF allowed for eISA to be used as a more sensitive alternative to other QTOF DIA and DDA approaches, and further, it enabled the acquisition of ESI TOF and ESI single quadrupole mass spectrometry instrumentation spectra with improved molecular identification confidence.

Application of a non-target workflow for the identification of specific contaminants using the example of the Nidda river basin

Non-target screening of water samples from the Nidda river basin in central Germany was conducted with the goal to identify previously unknown chemical contaminants and their emission sources. The focus was on organic, water-borne contaminants which were not typical to municipal wastewater. Grab samples of river water from 13 locations on the Nidda and 15 of its tributaries, in sum 112 samples, were analysed with high resolution LC-QToF-MS/MS. To facilitate the identification of substances, features originating from the same compound such as adducts and isotopologues as well as in-source fragments and species with multiple charge states were registered and grouped by a componentization step utilizing both retention times and peak shapes of the features to combine them in a single component. This led to a reduction of the number of features by an average of 1235 per sample (46%). These grouped features were prioritized if these were detected only in specific tributaries or specific river sections, reducing the number of components by an average of 913 per sample (78%). In addition, grouped features were labelled as typically found in municipal wastewater by combining data from 16 wastewater treatment plants located across Germany and Switzerland and comparing this to components detected in the Nidda basin. These were removed, leading to a further reduction of components by an average of 72 per sample (30%) for an average total reduction of 2536 per sample (93%). Finally, nine compounds, with emission sources in three specific tributaries, were identified, including the textile additive Nylostab S-EED®, which was previously not known to be an environmental contaminant, as well as naturally occurring compounds such as highly toxic microcystins.

Are (fluorinated) ionic liquids relevant environmental contaminants? High-resolution mass spectrometric screening for per- and polyfluoroalkyl substances in environmental water samples led to the detection of a fluorinated ionic liquid.

Fragmentation flagging (FF), a high-resolution mass spectrometric screening variant that utilizes intentionally produced indicative in-source fragments, was used to screen for per- and polyfluoroalkyl substances (PFASs) in surface waters. Besides expected legacy PFAS, FF enabled the detection of some rarely investigated representatives, such as trifluoromethanesulfonic acid (TFMSA). Additionally, a novel PFAS was detected and identified as tris(pentafluoroethyl)trifluorophosphate (FAP) via MS/MS experiments and confirmed with a reference standard. The first monitoring of FAP in 20 different surface waters revealed a localized contamination affecting three connected rivers with peak concentrations of up to 3.4μg/L. To the best of our knowledge, this is the first time FAP has been detected in environmental water samples. The detection of FAP, which is exclusively used as a constituent of ionic liquids (ILs), raises questions about the environmental relevance of ILs in general and particularly fluorinated ILs. A following comprehensive literature search revealed that ILs have already been intensely discussed as potential environmental contaminants, but findings reporting ILs in environmental (water) samples are almost non-existent. Furthermore, we address the relevance of ILs in the context of persistent, mobile, and toxic chemicals, which are at present gaining increasing scientific and regulatory interest, and as part of the PFAS "dark matter" that represents the gap between the amount of fluorine originating from known PFAS and the total adsorbable organically bound fluorine. Graphical abstract.

Revelation of Acyl Double Bond Positions on Fatty Acyl Coenzyme A Esters by MALDI/TOF Mass Spectrometry.

Fatty acyl coenzyme A esters (FA-CoAs) are important crossroad intermediates in lipid catabolism and anabolism, and the structures are complicated. Several mass spectrometric approaches have been previously described to elucidate their structures. However, a direct mass spectrometric approach toward a complete identification of the molecule, including the location of unsaturated bond(s) in the fatty acid chain has not been reported. In this study, we applied a simple MALDI/TOF mass spectrometric method to a near-complete characterization of long-chain FA-CoAs, including the location(s) of the double bond in the fatty acyl chain, and the common structural features that recognize FA-CoAs. Negative ion mass spectra of saturated, monounsaturated, and polyunsaturated FA-CoAs were acquired with a MALDI/TOF mass spectrometer using 2,5-dihydroxybenzoic acid as the matrix and ionized with a laser fluence two folds of the threshold to induce the in-source fragmentation (ISF) of the analytes. The resulting ISF spectra contained fragment ions arising from specific cleavages of the C-C bond immediate adjacent to the acyl double-bond. This structural feature affords locating the double-bond position(s) of the fatty acyl substituent. Thereby, positional isomer such as 18:3(n - 3) and 18:3(n - 6) FA-CoA can be differentiated without applying tandem mass spectrometry.

Unveiling the Fragmentation Mechanisms of Modified Amino Acids as the Key for Their Targeted Identification.

The alteration of modified amino acid (MAA) profiles in biological samples is related to important cellular, physiological, and pathological processes. To achieve the interpretation of their biochemical relevance, it is critical to define their whole chemical spectrum using metabolomic research works. We present a detailed in-source fragmentation (ISF) study based on the mechanisms of the major fragmentation reactions observed of diagnostic ions (DIs) generated in positive electrospray ionization for 57 amino acid standard compounds using capillary electrophoresis coupled with high-resolution mass spectrometry. The DIs presented and our in-house fragment library allowed us to establish a workflow for targeted extraction of MAAs. We present key examples showing successful findings such as the identification of N2-methyl-l-lysine, which provides insight into the lysine methylome. The experimental results presented prove that the use of ISF data, when combined with a thorough study of the fragmentation mechanisms, constitutes an informative source of accurate molecular identity.

Molecular characterization of polyethylene oxide based oligomers by surface-assisted laser desorption/ionization mass spectrometry using a through-hole alumina membrane as active substrate.

Molecular characterization of industrial oligomeric products is performed using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS), termed desorption ionization using a through-hole alumina membrane (DIUTHAME). This paper describes the unique feature of a DIUTHAME chip applying active SALDI, which generates specific types of fragments of polyglycol samples. Polyethylene oxide (PEO) and PEO-based materials were subjected to SALDI-MS. The influence of the presence or absence of a cationization salt on the mass spectrum was investigated. The resulting mass spectra composed of fragment ions were compared with those obtained by collision-induced dissociation (CID)-MS/MS. The specific fragment ions generated using the DIUTHAME chip were further subjected to high-energy CID-MS/MS. The addition of a cationization salt resulted in SALDI mass spectra with fewer fragment peaks. The mass spectra obtained without adding the cationization salt were composed of many more fragment ions caused by in-source decay. The fragmentation pattern was similar to that seen with low-energy CID. The resulting fragment ions were formed by selective cleavage at the C-O bond. High-energy CID-MS/MS can be performed for the specific fragment ions generated by in-source decay fragmentation. Molecular characterization of PEO-based oligomers by SALDI-MS using the DIUTHAME chip was successfully demonstrated. The selective fragmentation and high-energy CID-MS/MS of the in-source decay fragments made it possible to provide more detailed structural information. This unique feature of DIUTHAME gives it potential for use in new molecular characterization techniques.

Nucleobase derivatives induce in-source decay of oligonucleotides as new matrix-assisted laser desorption/ionization matrices.

For quality control of oligonucleotide therapeutics, accurate and efficient structural characterization using mass spectrometry techniques, such as liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), is essential. In MALDI MS analysis, matrix selection is critical and a new matrix could enable more efficient and rapid structural analysis. We hypothesized that nucleobase derivatives could act as matrices more efficiently than the currently used matrices for oligonucleotides because of structural similarity, which leads to close contact with the analyte. To evaluate their suitability as matrices, 16 nucleobase derivatives were selected and tested as matrix candidates for oligonucleotide analysis. Six of the 16 nucleobase derivatives acted as matrices for oligonucleotides. Particularly, 6-thioguanine (TG) performed well and induced clear in-source decay fragmentation. When TG or 2-amino-6-chloropurine was used as the matrix, oligonucleotides were ionized, and mainly the w and d fragment ions were observed. Herein we demonstrate that a 10-mer RNA or DNA sequence can be successfully characterized using TG as matrix and suggest the possibility of using nucleobase derivatives as novel matrices in oligonucleotide sequencing.

Influence of Lipid Fragmentation in the Data Analysis of Imaging Mass Spectrometry Experiments.

Imaging mass spectrometry (IMS) is becoming an essential technique in lipidomics. Still, many questions remain open, precluding it from achieving its full potential. Among them, identification of species directly from the tissue is of paramount importance. However, it is not an easy task, due to the abundance and variety of lipid species, their numerous fragmentation pathways, and the formation of a significant number of adducts, both with the matrix and with the cations present in the tissue. Here, we explore the fragmentation pathways of 17 lipid classes, demonstrating that in-source fragmentation hampers identification of some lipid species. Then, we analyze what type of adducts each class is more prone to form. Finally, we use that information together with data from on-tissue MS/MS and MS3 to refine the peak assignment in a real experiment over sections of human nevi, to demonstrate that statistical analysis of the data is significantly more robust if unwanted peaks due to fragmentation, matrix, and other species that only introduce noise in the analysis are excluded.

CROP: correlation-based reduction of feature multiplicities in untargeted metabolomic data.

Untargeted liquid chromatography-high-resolution mass spectrometry analysis produces a large number of features which correspond to the potential compounds in the sample that is analyzed. During the data processing, it is necessary to merge features associated with one compound to prevent multiplicities in the data and possible misidentification. The processing tools that are currently employed use complex algorithms to detect abundances, such as adducts or isotopes. However, most of them are not able to deal with unpredictable adducts and in-source fragments. We introduce a simple open-source R-script CROP based on Pearson pairwise correlations and retention time together with a graphical representation of the correlation network to remove these redundant features. The CROP R-script is available online at www.github.com/rendju/CROP under GNU GPL. Supplementary data are available at Bioinformatics online.

Nitrogen direct analysis in real time time-of-flight mass spectrometry (N2 DART-TOFMS) for rapid screening of forensic drugs.

Over the last ten years, helium direct analysis in real time time-of-flight mass spectrometry (He DART-TOFMS) has become an established technique in rapid screening of forensic drugs to decrease the time necessary to triage forensic drug cases, therefore contributing to backlog reduction and more timely criminal prosecution. Recently, we demonstrated that N2 DART was able to efficiently ionize all polar compounds except for a few extremely small ones such as methanol and acetonitrile. Therefore, N2 DART-TOFMS should be a suitable technique for rapid screening of forensic drugs. Nitrogen direct analysis in real time time-of-flight mass spectrometry (N2 DART-TOFMS) was performed using a JEOL AccuTOF mass spectrometer with an IonSense DART-100 ion source. A 3-min analytical protocol was used for the analysis of each sample. Sample introduction was accomplished by moving the closed end of a melting point capillary where approximately 1 μL sample solution was deposited or the exposed inside of a freshly cut tablet across the N2 gas stream between the DART-100 ion source and orifice 1 of the AccuTOF. Ten commonly abused drugs, eight synthetic cannabinoids and four controlled prescription drugs (CPDs) were analyzed. The limit of detection (LOD) was determined to be approximately 10 μg/mL or 10 pg in quantities. All drugs at the LOD level were positively identified using their [M + H]+ ions with mass errors less than 5 mDa. The identification were further supported by in-source fragment ions and characteristic N2 DART ions that are not commonly generated by He DART, e.g. [M + H + O]+ and [M + H + 2O]+ ions. It was concluded that the 3-min analytical protocol could be utilized in the analysis of seized drugs in the form of tablets and powders or prepared in solution. In consideration that N2 is readily available in the air and He is a non-renewable resource, N2 DART-TOFMS is a greener, cheaper and more convenient alternative to He DART-TOFMS in rapid screening of forensic drugs.

Atmospheric Solids Analysis Probe Coupled to a Portable Mass Spectrometer for Rapid Identification of Bulk Drug Seizures.

The emergence of ambient ionization techniques and their combination with smaller, cheaper mass spectrometers is beginning to make real the possibility of mass spectrometry measurements being made routinely outside of traditional laboratory settings. Here, we describe the development of an atmospheric solids analysis probe (ASAP) source for a commercially available miniaturized, single-quadrupole mass spectrometer and subsequent modification of the instrument to allow it to run as a deployable system; we further go on to describe the application of this instrument to the identification of the contents of drug seizures. For the drug seizure analysis, a small quantity of the material (powder, tablet, resin, etc.) was dissolved in ethanol and shaken to extract the analytes, the resulting solutions were then sampled by dipping a sealed glass capillary into the solution prior to analysis by ASAP-MS. Identification of the contents of the seizures was carried out using a NIST searching approach utilizing a bespoke spectral library containing 46 compounds representative of those most commonly encountered in UK forensic laboratories. In order to increase confidence in identification the library sample and subsequent analyses were carried out using a four-channel acquisition method; each channel in this method used a different cone voltage (15, 30, 50, and 70 V) inducing differing levels of in-source fragmentation in each channel; the match score across each channel was then used for identification. Using this developed method, a set of 50 real-life drug samples was analyzed with each of these being identified correctly using the library searching method.

Highly selective monitoring of in-source fragmentation sapogenin product ions in positive mode enabling group-target ginsenosides profiling and simultaneous identification of seven Panax herbal medicines

In-source fragmentation of ginsenosides in the positive ESI mode (pISF-G) frequently occurs, which results in little fragment information useful for the structural elucidation. We are aimed to unveil the genesic mechanism and explore its potential significance in quality control of Ginseng and the related compound formulae. By applying six high-resolution mass spectrometers from Agilent, Waters, and Thermo Fisher, we could primarily demonstrate the susceptibility of pISF-G. The ion clusters in the positive full-scan MS1 spectra were generated from the protonated sapogenins by successive elimination of H2O, and showed specificity for ginsenoside classification. Selective ion monitoring (SIM) of the sapogenin product ions could delineate group-target ginsenoside profiles from Ginseng. A high-selectivity characteristic chromatogram (CC) was elaborated for Ginseng, on the Vion™ IMS-QTOF mass spectrometer by IM (ion mobility) separation and quadrupole filtering of four sapogenin fragments (m/z 407.37/CCS 206.24 Å2; m/z 423.36/CCS 211.26 Å2; m/z 439.36/CCS 209.60 Å2; m/z 457.37/CCS 217.81 Å2). Chemometric analysis, based on the CC data of seven Ginseng drugs (P. ginseng, P. quinquefolius, P. notoginseng, Red ginseng, leaf of P. ginseng, P. japonicus, and P. japonicus var. major), disclosed 35 marker compounds. We could readily discriminate among P. ginseng, P. quinquefolius, and P. notoginseng, in 15 different compound formulae by identifying these marker compounds on both the Vion IMS-QTOF and QTrap 4500 mass spectrometers. Conclusively, SIM of the pISF-G sapogenin product ions renders a new concept of CC enabling the group-target profiling of ginsenosides and authentication of Ginseng and the related compound formulae.

Reactive metallocene cations as sensitive indicators of gas-phase oxygen and water.

Analysis of highly reactive compounds at very low concentration in solution using electrospray ionization mass spectrometry requires the use of exhaustively purified solvents. It has generally been assumed that desolvation gas purity needs to be similarly high, and so most chemists working in this space have relied upon high purity gas. However, the increasing competitiveness of nitrogen generators, which provide gas purity levels that vary inversely with flow rate, prompted an investigation of the effect of gas-phase oxygen on the speciation of ions. The most reactive species studied, the reduced titanium complex [Cp2Ti(NCMe)2]+[ZnCl3]- and the olefin polymerization pre-catalyst [Cp2Zr(μ-Me)2AlMe2]+[B(C6F5)4]-, only exhibited detectable oxidation when they were rendered coordinatively unsaturated through in-source fragmentation. Computational chemistry allowed us to find the most plausible pathways for the observed chemistry in the absence of observed intermediates. The results provide insight into the gas-phase oxidation or hydrolysis of these reactive species.

Distribution of Flavan-3-ol Species in Ripe Strawberry Fruit Revealed by Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging.

Flavan-3-ols, which comprise proanthocyanidins and their monomers, are major flavonoids in strawberries, and they have a wide range of biological activities and health benefits. However, their spatial distribution in strawberry fruit remains poorly understood. Therefore, we performed matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI), to visualize flavan-3-ols in ripe strawberry fruit. Peaks matching the m/z values of flavan-3-ols [M − H]− ions were detected in the negative ion mode using 1,5-diaminonaphthalene as matrix. Catechin and/or epicatechin, three B-type procyanidins, and two B-type propelargonidins were identified by MALDI-tandem MS. These flavan-3-ols were mainly distributed in the calyx, in and around the vascular bundles, and in the skin. In-source fragmentation of proanthocyanidins was determined using their standards, suggesting their distribution was mixed ion images of themselves, and fragment ions generated from those had a higher degree of polymerization. B-type procyanidins were predominantly distributed in the vascular bundles than in the skin, whereas B-type propelargonidins were almost equally distributed between the vascular bundles and skin, suggesting that their distribution patterns are different from the type of their flavan-3-ol monomers. Flavan-3-ols, especially B-type procyanidins, may help prevent pathogen infection not only in the skin but also in and around the vascular bundles.