Recent Developments in Machine Learning for Mass Spectrometry.

Statistical analysis and modeling of mass spectrometry (MS) data have a long and rich history with several modern MS-based applications using statistical and chemometric methods. Recently, machine learning (ML) has experienced a renaissance due to advents in computational hardware and the development of new algorithms for artificial neural networks (ANN) and deep learning architectures. Moreover, recent successes of new ANN and deep learning architectures in several areas of science, engineering, and society have further strengthened the ML field. Importantly, modern ML methods and architectures have enabled new approaches for tasks related to MS that are now widely adopted in several popular MS-based subdisciplines, such as mass spectrometry imaging and proteomics. Herein, we aim to provide an introductory summary of the practical aspects of ML methodology relevant to MS. Additionally, we seek to provide an up-to-date review of the most recent developments in ML integration with MS-based techniques while also providing critical insights into the future direction of the field.

Application of Laser-Induced Acoustic Desorption for Molecular Rotational Resonance Spectroscopy.

Molecular Rotational Resonance (MRR) spectroscopy is a uniquely precise tool for the determination of molecular structures of volatile compounds in mixtures, as the characteristic rotational transition frequencies of a molecule are extremely sensitive to its 3D structure through the moments of inertia in a three-dimensional coordinate system. This enables identification of the compounds based on just a few parameters that can be calculated, as opposed to, for example, mass spectrometric data, which often require expert analysis of 10-20 different signals and the use of many standards/model compounds. This paper introduces a new sampling technique for MRR, laser-induced acoustic desorption (LIAD), to allow the vaporization of nonvolatile and thermally labile analytes without the need for excessive heating or derivatization. In this proof-of-concept study, LIAD was successfully coupled to an MRR instrument to conduct measurements on seven compounds with differing polarities, molecular weights, and melting and boiling points. Identification of three isomers in a mixture was also successfully performed using LIAD/MRR. Based on these results, LIAD/MRR is demonstrated to provide a powerful approach for the identification of nonvolatile and/or thermally labile analytes with molecular weights up to 600 Da in simple mixtures, which does not require the use of reference compounds. In the future, applications to more complex mixtures, such as those relevant to pharmaceutical research, and quantitative aspects of LIAD/MRR will be reported.

Gas-Phase Reactivity of Quinoline-Based Singlet Oxenium Cations.

Isomeric quinolyloxenium cations were generated in the gas phase in an ion trap mass spectrometer to explore their reactions. The structures of some products were identified via collision-activated dissociation experiments involving model compounds to demonstrate that they have the expected heavy atom connectivity. The lack of radical reactions suggests that the cations have closed-shell singlet electronic ground states. Calculations (CASPT2/CASSCF(16,14)/cc-pVTZ//CASSCF(16,14)/cc-pVTZ) predict that their closed-shell singlet (1A') ground states are lower in energy by ca. 25 kcal mol-1 than their lowest-lying excited states. All cations are reactive toward dimethyl disulfide, dimethyl sulfide, and allyl iodide and most toward water and moderately reactive toward cyclohexane, reflecting their strongly electrophilic nature. They form adducts with nucleophiles in exothermic reactions (ca. 50 kcal mol-1 for dimethyl sulfide) that can fragment or be stabilized via IR emission. Most water adducts spontaneously isomerize to lower-energy tautomers. The nucleophiles preferentially add to those carbon atoms in the benzene ring that have the greatest positive charge (but not the carbonyl carbon). The cations react with cyclohexane via hydride abstraction by the oxygen atom. This is the only reaction that initially involves the oxygen atom and hence reflects the formally positively charged, monovalent oxygen atom in these cations.

Generation and reactivity of the fragment ion [B12I8S(CN)]- in the gas phase and on surfaces.

Gaseous fragment ions generated in mass spectrometers may be employed as "building blocks" for the synthesis of novel molecules on surfaces using ion soft-landing. A fundamental understanding of the reactivity of the fragment ions is required to control bond formation of deposited fragments in surface layers. The fragment ion [B12X11]- (X = halogen) is formed by collision-induced dissociation (CID) from the precursor [B12X12]2- dianion. [B12X11]- is highly reactive and ion soft-landing experiments have shown that this ion binds to the alkyl chains of organic molecules on surfaces. In this work we investigate whether specific modifications of the precursor ion affect the chemical properties of the fragment ions to such an extent that attachment to functional groups of organic molecules on surfaces occurs and binding of alkyl chains is prevented. Therefore, a halogen substituent was replaced by a thiocyanate substituent. CID of the precursor [B12I11(SCN)]2- ion preferentially yields the fragment ion [B12I8S(CN)]-, which shows significantly altered reactivity compared to the fragment ions of [B12I12]2-. [B12I8S(CN)]- has a previously unknown structural element, wherein a sulfur atom bridges three boron atoms. Gas-phase reactions with different neutral reactants (cyclohexane, dimethyl sulfide, and dimethyl amine) accompanied by theoretical studies indicate that [B12I8S(CN)]- binds with higher selectivity to functional groups of organic molecules than fragment ions of [B12I12]2- (e.g., [B12I11]- and [B12I9]-). These findings were further confirmed by ion soft-landing experiments, which showed that [B12I8S(CN)]- ions attacked ester groups of adipates and phthalates, whereas [B12I11]- ions only bound to alkyl chains of the same reagents.

Characterization of the degradation products of lignocellulosic biomass by using tandem mass spectrometry experiments, model compounds, and quantum chemical calculations.

Biomass-derived degraded lignin and cellulose serve as possible alternatives to fossil fuels for energy and chemical resources. Fast pyrolysis of lignocellulosic biomass generates bio-oil that needs further refinement. However, as pyrolysis causes massive degradation to lignin and cellulose, this process produces very complex mixtures. The same applies to degradation methods other than fast pyrolysis. The ability to identify the degradation products of lignocellulosic biomass is of great importance to be able to optimize methodologies for the conversion of these mixtures to transportation fuels and valuable chemicals. Studies utilizing tandem mass spectrometry have provided invaluable, molecular-level information regarding the identities of compounds in degraded biomass. This review focuses on the molecular-level characterization of fast pyrolysis and other degradation products of lignin and cellulose via tandem mass spectrometry based on collision-activated dissociation (CAD). Many studies discussed here used model compounds to better understand both the ionization chemistry of the degradation products of lignin and cellulose and their ions' CAD reactions in mass spectrometers to develop methods for the structural characterization of the degradation products of lignocellulosic biomass. Further, model compound studies were also carried out to delineate the mechanisms of the fast pyrolysis reactions of lignocellulosic biomass. The above knowledge was used to assign likely structures to many degradation products of lignocellulosic biomass.

Molecular Precursor Approach to Sulfur-Free CuInSe2: Replacing Thiol Coordination in Soluble Metal Complexes.

Solution-processed CuInSe2 films have generally relied on sulfide or sulfoselenide precursor films that, during the grain growth process, hamper the growth of thicker films and lead to the formation of a fine-grain layer. However, recent research has indicated that sulfur reduction in the precursor film modifies the grain growth mechanism and may enable the fabrication of thicker absorbers that are free of any fine-grain layer. In this work, we pursue direct solution deposition of sulfur-free CuInSe2 films from the molecular precursor approach. To this end, we tune the amine-thiol reactive solvent system and study the changes to the resulting soluble complexes through a combination of analytical techniques. We show that by reactively dissolving indium(III) selenide and selenium in solutions of n-butylamine and 1,2-ethanedithiol, a metal thiolate species is formed, and that this metal thiolate can be modified by isolation from the thiol-containing solvent via precipitation. As the quantity of selenium in the ink increases, the thiol content in the complex decreases, eventually producing soluble [InSex]- species. Extending this method to be used with copper selenide as a copper source, molecular precursor inks can be made for solution-processed, sulfur-free CuInSe2 films. We then show that these CuInSe2 precursor films can be fully coarsened without a fine-grain layer formation, even at the desired thicknesses of 2 µm and greater.

Real-Time Mass Spectrometric Detection of Reaction Intermediates Formed during Laser-Induced UV/H2O2 Advanced Oxidation of 2-Methylbenzoisothiazol-3-one.

Knowledge on short-lived reaction intermediates is often essential for mechanistic investigations of organic reactions and for reaction optimization. Unfortunately, most conventional analytical methods are too slow to allow the detection of short-lived reaction intermediates. Herein, a direct laser desorption/ionization method coupled with linear quadrupole ion trap/orbitrap high-resolution tandem mass spectrometry was used for the detection and structural characterization of several previously proposed but undetected reaction intermediates formed during laser-induced UV/H2O2 advanced oxidation of 2-methylbenzoisothiazol-3-one. The elemental compositions of most detected (ionized) compounds were determined. Tandem mass spectrometry experiments based on gas-phase collision-activated dissociation (CAD) were conducted to gain information on the ion structures. The mechanisms of the CAD reactions were explored using high-level quantum chemical calculations to support the structures proposed for the neutral reaction intermediates formed during the laser-induced UV/H2O2 advanced oxidation of 2-methylbenzoisothiazol-3-one. In the negative-ion mode experiments, anions corresponding to three reaction intermediates were detected and structurally characterized: 1-hydroxy-2-methyl-1,2-dihydro-3H-1λ4-benzo[d]isothiazol-3-one, 2-(methylcarbamoyl)benzenesulfinic acid, and 2-(dihydroxy(oxo)-λ6-sulfaneyl)-N-methylbenzamide. One of the final products, 2-(methylcarbamoyl)benzenesulfonic acid, was also detected and characterized. In positive-ion mode experiments, cations corresponding to the reactant, 2-methylbenzoisothiazol-3-one, as well as an intermediate reaction product and the two final reaction products, 2-methylbenzo[d]isothiazol-3(2H)-one 1-oxide, N-methylsaccharine, and 2-(methylcarbamoyl)benzenesulfonic acid, respectively, were detected and identified. This research substantially improved the understanding on the reaction intermediates formed during laser-induced UV/H2O2 advanced oxidation of 2-methylbenzoisothiazol-3-one, which facilitates the delineation of the reaction mechanisms occurring in these processes.

Differentiation of the Aziridine Functionality from Related Functional Groups in Protonated Analytes by Using Selective Ion-Molecule Reactions Followed by Collision-Activated Dissociation in a Linear Quadrupole Ion Trap Mass Spectrometer.

Aziridines are commonly used as reagents for the synthesis of drug substances although they are potentially mutagenic and genotoxic. Therefore, their unambiguous detection is critically important. Unfortunately, tandem mass spectrometry (MS2) based on collision-activated dissociation (CAD), a powerful method used for the identification of many unknown compounds in complex mixtures, does not provide diagnostic fragmentation patterns for ionized aziridines. Therefore, a different mass spectrometry approach based on MS3 experiments is presented here for the identification of the aziridine functionalities. This approach is based on selective gas-phase ion-molecule reactions of protonated analytes with tris(dimethylamino)borane (TDMAB) followed by diagnostic CAD reactions in a modified linear quadrupole ion trap (LQIT) mass spectrometer. TDMAB reacts with protonated aziridines by forming adduct ions that have lost a dimethylamine (DMA) molecule ([M + H + TDMAB - HN(CH3)2]+). CAD on these product ions generated diagnostic fragment ions with m/z-values 25- and 43-units lower than those of the ion-molecule reaction product ions. None of the ion-molecule reaction product ions formed from other, structurally related, protonated analytes produced related fragment ions. Quantum chemical calculations were employed to explore the mechanisms of the observed reactions.

Modification of a Quadrupole/Orbitrap/Linear Quadrupole Ion Trap Tribrid Mass Spectrometer for Diagnostic Gas-Phase Ion-Molecule Reactions.

Tandem mass spectrometry based on diagnostic gas-phase ion-molecule reactions represents a robust method for functional group identification in unknown compounds. To date, most of these reactions have been studied using unit-resolution instruments, such as linear quadrupole ion traps and triple quadrupoles, which cannot be used to obtain elemental composition information for the species of interest. In this study, a high-resolution mass spectrometer, a quadrupole/orbitrap/linear quadrupole ion trap tribrid, was modified by installing a portable reagent inlet system to obtain high-resolution data for ion-molecule reactions. Examination of a previously published test system, the reaction between protonated 1,1'-sulfonyldiimizadole with 2-methoxypropene, demonstrated the ability to perform ion-molecule reactions on the modified tribrid mass spectrometer. High-resolution data were obtained for ion-molecule reactions of three isobaric ions (protonated glycylalanine, protonated glutamine, and protonated lysine) with diethylmethoxyborane. On the basis of these data, the isobaric ions can be differentiated based on both their measured accurate mass as well as the different product ions they generated upon the ion-molecule reactions. In a different experiment, analyte ions were subjected to collision-induced dissociation (CID), and the structures of the resulting fragment ions were examined via diagnostic ion-molecule reactions. This experiment allows for the functional group interrogation of fragment ions and can be used to improve the understanding of the structures of fragment ions generated in the gas phase.

Mechanistic Study into Free Radical-Activated Glycan Dissociations through Isotope-Labeled Cellobioses.

Inspired by the electron-activated dissociation technique, the most potent tool for glycan characterization, we recently developed free radical reagents for glycan structural elucidation. However, the underlying mechanisms of free radical-induced glycan dissociation remain unclear and, therefore, hinder the rational optimization of the free radical reagents and the interpretation of tandem mass spectra, especially the accurate assignment of the relatively low-abundant but information-rich ions. In this work, we selectively incorporate the 13C and/or 18O isotopes into cellobiose to study the mechanisms for free radical-induced dissociation of glycans. The eight isotope-labeled cellobioses include 1-13C, 3-13C, 1'-13C, 2'-13C, 3'-13C, 4'-13C, 5'-13C, and 1'-13C-4-18O-cellobioses. Upon one-step collisional activation, cross-ring (X ions), glycosidic bond (Y-, Z-, and B-related ions), and combinational (Y1 + 0,4X0 ion) cleavages are generated. These fragment ions can be unambiguously assigned and confirmed by the mass difference of isotope labeling. Importantly, the relatively low-abundant but information-rich ions, such as 1,5X0 + H, 1,4X0 + H, 2,4X0 + H-OH, Y1 + 0,4X0, 2,5X1-H, 3,5X0-H, 0,3X0-H, 1,4X0-H, and B2-3H, are confidently assigned. The mechanisms for the formations of these ions are investigated and supported by quantum chemical calculations. These ions are generally initiated by hydrogen abstraction followed by sequential ß-elimination and/or radical migration. Here, the mechanistic study for free radical-induced glycan dissociation allows us to interpret all of the free radical-induced fragment ions accurately and, therefore, enables the differentiation of stereochemical isomers. Moreover, it provides fundamental knowledge for the subsequent development of bioinformatics tools to interpret the complex free radical-induced glycan spectra.

Determination of the compound class and functional groups in protonated analytes via diagnostic gas-phase ion-molecule reactions.

Diagnostic gas-phase ion-molecule reactions serve as a powerful alternative to collision-activated dissociation for the structural elucidation of analytes when using tandem mass spectrometry. The use of such ion-molecule reactions has been demonstrated to provide a robust tool for the identification of specific functional groups in unknown ionized analytes, differentiation of isomeric ions, and classification of unknown ions into different compound classes. During the past several years, considerable efforts have been dedicated to exploring various reagents and reagent inlet systems for functional-group selective ion-molecule reactions with protonated analytes. This review provides a comprehensive coverage of literature since 2006 on general and predictable functional-group selective ion-molecule reactions of protonated analytes, including simple monofunctional and complex polyfunctional analytes, whose mechanisms have been explored computationally. Detection limits for experiments involving high-performance liquid chromatography coupled with tandem mass spectrometry based on ion-molecule reactions and the application of machine learning to predict diagnostic ion-molecule reactions are also discussed.

Differentiation of Seven Isomeric n-Pentylquinoline Radical Cations Based on Energy-Resolved Medium-Energy Collision-Activated Dissociation.

Asphaltenes, a major and undesirable component of heavy crude oil, contain many different types of large aromatic compounds. These compounds include nitrogen-containing heteroaromatic compounds that are thought to be the main culprit in the deactivation of catalysts in crude oil refinery processes. Unfortunately, prevention of this is challenging as the structures and properties of the nitrogen-containing heteroaromatic compounds are poorly understood. To facilitate their structural characterization, an approach based on ion-trap collision-activated dissociation (ITCAD) tandem mass spectrometry followed by energy-resolved medium-energy collision-activated dissociation (ER-MCAD) was developed for the differentiation of seven isomeric molecular radical cations of n-pentylquinoline. The fragmentation of each isomer was found to be distinctly different and depended largely on the site of the alkyl side chain in the quinoline ring. In order to better understand the observed fragmentation pathways, mechanisms for the formation of several fragment ions were delineated based on quantum chemical calculations. The fast benzylic α-bond cleavage that dominates the fragmentation of analogous nonheteroaromatic alkylbenzenes was only observed for the 3-isomer as the major pathway due to the lack of favorable low-energy rearrangement reactions. All the other isomeric ions underwent substantially lower-energy rearrangement reactions as their alkyl chains were found to interact mostly via 6-membered transition states either with the quinoline nitrogen (2- and 8-isomers) or the adjacent carbon atom in the quinoline core (4-, 5-, 6-, and 7-isomers), which lowered the activation energies of the fragmentation reactions. The presented analytical approach will facilitate the structural characterization of nitrogen-containing heteroaromatic compounds in asphaltenes.

Detection of the tetrahedral reaction intermediate of the reaction of acetyl chloride with ethanol in microdroplets via laser desorption/ionization tandem mass spectrometry.

Understanding the fundamental mechanisms of chemical reactions is of great interest to scientists working in many fields as it enables the rationalization, prediction, and design of reactions. Many chemical processes involve the formation of short-lived reaction intermediates, most of which cannot be isolated and are challenging to detect. One such intermediate is the tetrahedral intermediate often proposed to be generated upon the reactions of acetyl chlorides with simple alcohols via an addition/elimination mechanism. However, the formation of this tetrahedral intermediate is a subject of controversy as it has not been detected. Furthermore, some kinetic evidence suggests the SN2 mechanism for this reaction. In the present investigation, a 266 nm pulsed Nd:YAG laser was used to evaporate and ionize reactants, reaction intermediates, and products in microdroplets of acetyl chloride and ethanol. A linear quadrupole ion trap mass spectrometer was used to detect the ions and collision-activated dissociation (CAD) experiments were employed for their structural characterization. The results demonstrate the formation of the protonated tetrahedral intermediate of the addition/elimination reaction. The protonated reaction intermediate was isolated and subjected to CAD, which resulted in the loss of water and ethylene, thus confirming its structure. These results demonstrate that the ethanolysis of acetyl chloride proceeds via an addition/elimination mechanism involving a tetrahedral reaction intermediate. However, the parallel occurrence of the SN2 mechanism cannot be ruled out.

Proton Affinities of Alkanes.

Chemical characterization of complex mixtures of large alkanes is critically important for many fields, including petroleomics and the development of renewable transportation fuels. Tandem mass spectrometry is the only analytical method that can be used to characterize such mixtures at the molecular level. Many ionization methods used in mass spectrometry involve proton transfer to the analyte. Unfortunately, very few proton affinity (PA) values are available for alkanes. Indeed, previous research has shown that most protonated alkanes (MH+) are not stable but fragment spontaneously via the elimination of a hydrogen molecule to form [M - H]+ ions. Here, the PAs of several n-alkanes and alkylcyclohexanes containing 5-8 carbon atoms, n-pentane, n-hexane, n-heptane, n-octane, cyclohexane, methylcyclohexane, and ethylcyclohexane, were determined via bracketing experiments by using a linear quadrupole ion trap mass spectrometer. Monitoring the formation of the [M - H]+ ions in reactions between the alkanes and protonated reference bases with known PAs revealed that the PAs of all the alkanes fell into the range 721 ± 20 kJ mol-1. In order to obtain a more accurate estimate of the relative PAs of different alkanes, two alkanes were introduced simultaneously into the ion trap and allowed to react with the same protonated reference base. Based on these experiments, the longer the alkyl chain in an n-alkane or alkylcyclohexane the greater the PA. Further, when considering alkanes with the same number of carbon atoms, the PAs of those with a cyclohexane ring were found to be greater than those with no such ring.

Binding of saturated and unsaturated C6-hydrocarbons to the electrophilic anion [B12Br11]-: a systematic mechanistic study.

The highly reactive gaseous ion [B12Br11]- is a metal-free closed-shell anion which spontaneously forms covalent bonds with hydrocarbon molecules, including alkanes. Herein, we systematically investigate the reaction mechanism for binding of [B12Br11]- to the five hexane isomers yielding [B12Br11(C6H14)]-, as well as to cyclohexane and several hexene isomers (yielding [B12Br11(C6H12)]-) using collision-induced dissociation (CID), infrared photodissociation spectroscopy (IRPD) and computational methods. CID of the different [B12Br11(C6H14)]- ions results in distinct fragmentation patterns dependent on the structure of the hexane isomer. The observed fragmentation reactions provide insights into the addition mechanism of [B12Br11]- to hexane. Based on the observed CID patterns, we identified that either B-C bond formation through heterolytic C-C or C-H bond cleavages or B-H bond formation through heterolytic C-H cleavage occur dependent on the structure of the hexane isomer. Meanwhile, we observe identical CID spectra of adducts originating from isomers of C6H12. Spectroscopic investigations of adducts of 1-hexene and cyclohexane indicate the same product structure with an open C6 chain. Computational investigations evidenced that low lying transition states are present, which enable a ring opening reaction of cyclohexane when binding to [B12Br11]-.

A Diagnostic Nitrosamine Detection Approach for Pharmaceuticals by Using Tandem Mass Spectrometry Based on Diagnostic Gas-Phase Ion-Molecule Reactions.

N-Nitrosamines are strictly regulated in pharmaceutical products due to their carcinogenic nature. Therefore, the ability to rapidly and reliably identify the N-nitroso functionality is urgently needed. Unfortunately, not all ionized N-nitroso compounds produce diagnostic fragment ions and hence tandem mass spectrometry based on collision-activated dissociation (CAD) cannot be used to consistently identify the N-nitroso functionality. Therefore, a more reliable method was developed based on diagnostic functional-group selective ion-molecule reactions in a linear quadrupole ion trap mass spectrometer. 2-Methoxypropene (MOP) was identified as a reagent that reacts with protonated N-nitrosamines in a diagnostic manner by forming an adduct followed by the elimination of 2-propenol (CH3C(OH)═CH2]). From 18 protonated N-nitrosamine model compounds studied, 15 formed the diagnostic product ion. The lack of the diagnostic reaction for three of the N-nitrosamine model compounds was rationalized based on the presence of a pyridine ring that gets preferentially protonated instead of the N-nitroso functionality. These N-nitrosamines can be identified by subjecting a stable adduct formed upon ion-molecule reactions with MOP to CAD. Further, the ability to use ion-molecule reactions followed by CAD to differentiate protonated O-nitroso compounds with a pyridine ring from analogous N-nitrosamines was demonstrated This methodology is considered to be robust for the identification of the N-nitroso functionality in unknown analytes. Lastly, HPLC/MS2 experiments were performed to determine the detection limit for five FDA regulated N-nitrosamines.

A Portable Reagent Inlet System Designed to Diminish the Impact of Air and Water to Ion-Molecule Reactions Studied in a Linear Quadrupole Ion Trap.

A portable reagent inlet system for a linear quadrupole ion trap (LQIT) mass spectrometer was designed to diminish the impact of air and water on gas-phase ion-molecule reactions. Compared to the traditional reagent mixing manifolds that has been extensively used for decades, the portable system is much simpler and has fewer junctions and a smaller inner space. These changes reduce the amount of air and water introduced into the mass spectrometer with the reagent. Furthermore, unlike the traditional manifolds, the portable system can be easily attached to or detached from the LQIT mass spectrometer. Finally, the price of the portable system is only 1/10 of that of a traditional manifold as estimated in 2022. Therefore, the portable system has several advantages over the traditional reagent mixing manifolds.

Gas-Phase Reactivity of Phenylcarbyne Anions.

Gas-phase reactivities of the phenylcarbyne anion and its four derivatives were studied using a linear quadrupole ion trap mass spectrometer. The phenylcarbyne anions were calculated to have a triplet ground state (singlet-triplet splittings of 4-9 kcal mol-1), with the exception of the 4-cyanophenylcarbyne anion that has a singlet ground state (singlet-triplet splitting of -1.9 kcal mol-1). Only the phenylcarbyne anions with a triplet ground state react with acetone and dimethyl disulfide via radical mechanisms. On the other hand, only the phenylcarbyne anion with a singlet ground state abstracts H2O and H2C═C═O from acetic acid via electrophilic addition of the reagents to the anion. Finally, two hydroxy-substituted phenylcarbyne anions (with triplet ground states) partially tautomerize with the assistance of reagent molecules to the more stable distonic phenylcarbene anions. This occurs via abstraction of a proton from the reagent by the phenylcarbyne anion to generate a neutral (triplet) phenylcarbene and a reagent anion, which is followed by proton abstraction from the hydroxyl group of the neutral phenylcarbene by the reagent anion to generate the distonic phenylcarbene anion in an excited triplet state. Experiments performed on deuterated hydroxy-substituted phenylcarbyne anions verified the mechanism. The reactivities of the distonic phenylcarbene anions were found to be quite different from those of the phenylcarbyne anions. For example, they were found to abstract CH2 from acetonitrile, which is initiated by C-H insertion─typical singlet carbene reactivity.

Differentiation of Protonated Sulfonate Esters from Isomeric Sulfite Esters and Sulfones by Gas-Phase Ion-Molecule Reactions Followed by Diagnostic Collision-Activated Dissociation in Tandem Mass Spectrometry Experiments.

Sulfonate esters, a class of potentially mutagenic drug impurities, are strictly regulated in pharmaceuticals. On the other hand, sulfite esters and sulfones, analogs of sulfonate esters, have limited safety concerns. However, previously developed analytical methods for sulfonate ester identification cannot be used to differentiate sulfonate esters from the isomeric sulfite esters and sulfones. A tandem mass spectrometric method is introduced here for the differentiation of these compounds. Diisopropoxymethylborane (DIMB) reacts with protonated sulfonate esters, sulfite esters, and sulfones (and many other compounds) in the gas phase to form the product ion [M + H + DIMB - CH3CH(OH)CH3]+. Upon collision-activated dissociation (CAD), these product ions generate diagnostic fragment ions that enable the differentiation of sulfonate esters, sulfite esters, and sulfones from each other. For example, SO2 elimination enabled the unambiguous identification of sulfite esters. On the other hand, elimination of CH3B═O followed by elimination of (CH3)2C═O was only observed for sulfonate esters. Neither type of diagnostic fragment ions was detected for the products of sulfones. However, the product ions formed for sulfones with an additional hydroxyl substituent underwent the elimination of another CH3CH(OH)CH3 molecule, which enabled their identification. Finally, ion-molecule reactions of DIMB with various other functionalities were also examined. Some of them yielded the product ions [M + H + DIMB - CH3CH(OH)CH3]+ but none of these product ions underwent the diagnostic CAD reactions discussed above. Quantum chemical calculations were employed to explore the mechanisms of the reactions. The limits of detection for the diagnostic ion-molecule reaction product ions in high-performance liquid chromatography (HPLC)/mass spectrometry (MS2) experiments were found to range from 0.075 to 1.25 nmol.