An intriguing "reversible reaction" in the fragmentation of deprotonated dicamba and benzoic acid in a Q-orbitrap mass spectrometer: Loss and addition of carbon dioxide.

RATIONALE: Loss of carbon dioxide is an important characteristic fragmentation reaction of deprotonated benzoic acid and its derivatives in electrospray ionization mass spectrometry. However, researchers have rarely noticed or believed that the loss of carbon dioxide in multistage mass spectrometry is a "reversible reaction," that is, the fragment anion generated by carbon dioxide loss can capture another carbon dioxide to regenerate its precursor ion. METHODS: The fragmentation of the [M - H]- ions of dicamba (3,6-dichloro-2-methoxybenzoic acid) and benzoic acid was performed with an electrospray ionization hybrid quadrupole-orbitrap mass spectrometer. The structural confirmation of the precursor ions and their product ions was supported by accurate mass (elemental composition) analysis. Pseudo-MS3 experiments (in-source collision-induced dissociation as MS2 ) and isotope labelling experiments were used to confirm the addition of carbon dioxide to the product ions in MS2 . RESULTS: In the fragmentation of deprotonated dicamba (m/z 219), the relative abundance of the precursor ion does not decrease significantly or even increases as the collision energy increases. When the m/z 145 and 175 product ions were isolated in the mass analyzer, the ions 44 m/z units larger (m/z 189 and 219) were generated spontaneously, indicating the formation of carbon dioxide adduct ions. In the fragmentation of deprotonated [carboxyl-13 C]-benzoic acid (m/z 122), a deprotonated [carboxyl-12 C]-benzoic acid ion (m/z 121) was generated which was derived from 13 CO2 loss and 12 CO2 addition. The isotope labelling experiment further supports the formation of CO2 -attached ions in the fragmentation of deprotonated benzoic acids. CONCLUSIONS: Under collisional activation, deprotonated dicamba and benzoic acids easily undergo carbon dioxide loss, but the decarboxylated product anions have an appropriate nucleophilicity to carbon dioxide and they can capture a background carbon dioxide molecule remaining in the vacuum system to regenerate the precursor ions. This study provides a new and deeper understanding of the gas-phase chemistry of deprotonated benzoic acid derivatives in mass spectrometry.

Identification of new interferences leached from plastic microcentrifuge tubes in electrospray ionization mass spectrometry.

RATIONALE: The incredible sensitivity of the modern mass spectrometry instrument enables scientists to detect a large number of molecules ranging from small organic compounds to biological macromolecules. However, the same sensitivity often throws up challenges with respect to background interferences and contaminants. The identification and source of these contaminants is very important for reducing background contamination and ensuring the accuracy of the analysis results. METHODS: The interfering compounds were analyzed by high-performance liquid chromatography coupled with a hybrid quadrupole-orbitrap mass spectrometer. The structural analysis was conducted by obtaining the accurate masses of precursors and their fragment ions. The retention time and MS/MS spectrum of one of the interfering compounds (N-lauryldiethanolamine) were compared with an authentic standard to reach an unequivocal structural assignment. RESULTS: The interferences (m/z 274 and 318 in positive mode) were observed during the analysis of herbicides in tea samples by electrospray ionization mass spectrometry (ESI-MS). Their structures were identified to be N-lauryldiethanolamine and N-(2-hydroxyethyl)-N-(2-(2-hydroxyethoxy)ethyl)dodecylamine by fragmentation interpretation and further confirmed by a standard compound. These interferences were found to be leached from the plastic microcentrifuge tubes used during sample pretreatment. The plastic tubes from two of the five suppliers tested were found to contain these two interferences. CONCLUSIONS: In this work, we presented an example about the observation, identification and source of interferences in ESI-MS. The N-lauryldiethanolamine and other ethoxylated aliphatic alkylamines are common plastic antistatic agents. They possess high proton affinity so that they show a strong response in ESI positive mode. In order to avoid their interference during mass spectrometric analysis we need to choose plastic tubes (or other plastic materials) that do not contain such antistatic agents.

A method based on precolumn derivatization and ultra high performance liquid chromatography with high-resolution mass spectrometry for the simultaneous determination of phthalimide and phthalic acid in tea.

Phthalimide can be formed from either the degradation of folpet and phosmet, or reaction of phthalic anhydride with primary amino groups. Consequently, the sum of phthalimide and folpet, expressed as folpet-residue definition, is highly prone to false-positive levels of folpet in tea. An analytical method is thus urgently needed to investigate the residue level and source of phthalimide in tea. In this work, we developed an accurate method of determining phthalimide and phthalic acid (the indicator of phthalic anhydride) by acetonitrile extraction and 3-bromopropyltrimethylammonium bromide derivatization coupled with ultra high performance liquid chromatography and high-resolution mass spectrometry. The method was validated, and linearity (correlation coefficients > 0.99) was obtained. Satisfactory recoveries at 10, 20, 50, and 100 µg/kg ranged from 76 to 117%, and the intra- and interday accuracies were <23%. The limit of quantification for phthalimide and phthalic acid was 10 µg/kg. The developed method was further successfully used to determine phthalimide and phthalic acid in some tea samples. The positive rate of phthalimide and phthalic acid detected in the tea samples ranged from 30-75 and 50-90%, respectively.

Neutral losses of sodium benzoate and benzoic acid in the fragmentation of the [M + Na]+ ions of methoxyfenozide and tebufenozide via intramolecular rearrangement in electrospray ionization tandem mass spectrometry.

RATIONALE: Electrospray ionization (ESI) tandem mass spectrometry can be applied to determine structural information about organic compounds. The [M + Na]+ ion is one of the major precursor ions in ESI mass spectrometry, but its fragmentation mechanism study is still insufficient. This study reveals the interesting fragmentation reactions of the [M + Na]+ ions of methoxyfenozide and tebufenozide. METHODS: The fragmentations of the [M + Na]+ , [M + Li]+ , and [M + H]+ ions of methoxyfenozide and tebufenozide were studied using a hybrid quadrupole-orbitrap mass spectrometer and an ion trap mass spectrometer. A hydrogen/deuterium (H/D)-exchange experiment in the amide group of methoxyfenozide allowed for the confirmation of the fragmentation mechanism. Density functional theory (DFT) calculations were performed for a further understanding of the fragmentation mechanism of the [M + Na]+ ion of methoxyfenozide. RESULTS: Neutral losses of sodium benzoate and benzoic acid in the fragmentation of the [M + Na]+ ions of methoxyfenozide and tebufenozide were observed as the major fragmentation pathways. In contrast, similar fragmentations were not observed or minor pathways in the fragmentation of the [M + Li]+ and [M + H]+ ions of methoxyfenozide and tebufenozide. In addition, a minor product ion resulting from loss of NaOH was identified, which was the first reported example in the fragmentation of sodiated compounds in mass spectrometry. CONCLUSIONS: Losses of sodium benzoate and benzoic acid in the fragmentation of the [M + Na]+ ions of methoxyfenozide and tebufenozide are proposed to be formed through an intramolecular rearrangement reaction, which is supported by DFT calculations. An H/D-exchange experiment confirms that the carboxyl hydrogen of benzoic acid and the hydrogen of NaOH exclusively derive from the amide hydrogen of the precursor ion. This study enriches our knowledge on the Na+ -induced fragmentation reactions. Copyright © 2016 John Wiley & Sons, Ltd.

Doubly charged trimeric cluster ions: effective in mutual chiral recognition of tadalafil and three proton pump inhibitors.

Mutual chiral recognition of four stereoisomers of tadalafil and three pairs of enantiomers of proton pump inhibitors (PPIs) including pantoprazole, lansoprazole, and omeprazole, as well as quantitative analysis of enantiomeric excess is achieved on the basis of the competitive fragmentation of doubly charged trimeric NiII cluster ions. Compared with a singly charged trimeric cluster ion, a doubly charged trimeric cluster ion was proved efficient in the recognition of chiral drugs with one or multiple chiral centers, due to its rich fragmentation ions. Upon collision-induced dissociation (CID), the cluster ion [NiII(PPIs)(tadalafil)2]2+ yielded two diagnostic ions [tadalafil + H]+ and [tadalafil - benzo[d][1,3]dixoloe]+ through electrospray ionization quadrupole time-of-flight mass spectrometry. The abundance ratio of the two fragment ions relied mainly on the configuration of PPIs and tadalafil, and therefore the chiral selectivity (Rchiral) of one enantiomer relative to the others is different. The chiral recognition of all four stereoisomers of tadalafil was achieved by using S configuration PPIs as references, and S-omeprazole showed the best selectivity. The Rchiral values for R,R/S,S, R,S/S,R, R,R/R,S and R,R/S,R-tadalafils were 1.47, 1.17, 2.37, and 2.10, respectively. In a reciprocal process, the Rchiral was 1.36 and 1.31 for R/S-pantoprazole and R/S-lansoprazole, respectively, by using R,R-tadalafil as a reference. Although omeprazole is a racemic drug, it can also be discriminated with S-omeprazole. Calibration curves were constructed with favorable correlation coefficients (r2 > 0.991) by relating the ln(Rchiral) values to the isomeric composition in a mixture. The sensitivity of the methodology allows mixtures to be analyzed for the enantiomeric excess (ee) by recording the ratios of fragment ion abundances in a mass spectrum. The sensitivity of the methodology allowed the determination of enantiomeric impurities of 5% molar composition in individual compounds present in mixtures; the diastereoisomeric impurity of R,R-tadalafil could be quantified even at 1%. We believe that the developed method not only has scientific significance in qualitative and quantitative chiral analyses of tadalafil and PPIs, but also provides great opportunity for enabling the discrimination on a wide range of chiral drugs.

Gas-phase Smiles rearrangement reactions of deprotonated N-phenylbenzamides studied by electrospray ionization tandem mass spectrometry.

RATIONALE: Electrospray ionization tandem mass spectrometry (ESI-MS(n)) is an invaluable tool for the study of gas-phase reactions. When N-phenylbenzamide is analyzed in negative ion mode, the nucleophilic deprotonated site of nitrogen or oxygen, together with the adjacent electrophilic phenyl carbon in the same molecule, provides a useful opportunity to study the intramolecular nucleophilic reaction in the gas phase. METHODS: All MS(n) experiments of deprotonated N-phenylbenzamides were conducted on an ion trap mass spectrometer using ESI in negative ion mode. The accurate masses of fragments were measured on an ESI quadrupole time-of-flight mass spectrometer in negative ion mode. Theoretical calculations were conducted at the B3LYP/6-31++G(d,p) level of density functional theory using the Gaussian 03 program. RESULTS: When the polarity of the substituent on the aniline ring was changed, gas-phase Smiles rearrangement reactions could be initiated by different atoms in the anionic center. Upon collisional activation, loss of CO from deprotonated N-phenylbenzamides could be observed, which can be interpreted as a nitrogen anion triggering the Smiles rearrangement reaction through a three-membered ring transition state. As the aniline ring was substituted by a strong electron-withdrawing group (e.g., NO(2), COCH(3), or CF(3)) at the para position, a characteristic phenolate anion was obtained, which was derived from the Smiles rearrangement reaction initiated by the oxygen anion through a four-membered ring transition state. CONCLUSIONS: In the fragmentation of deprotonated N-phenylbenzamides, the gas-phase Smiles rearrangement reaction initiated by either the nitrogen or the oxygen atom can proceed. The findings in this study have not only enriched knowledge on the gas-phase Smiles rearrangement reactions, but also provided valuable information for understanding the rearrangements of deprotonated aromatic amides in gas phase.

Benzyl anion transfer in the fragmentation of N-(phenylsulfonyl)-benzeneacetamides: a gas-phase intramolecular S(N)Ar reaction.

In this study, we report a gas-phase benzyl anion transfer via intramolecular aromatic nucleophilic substitution (SNAr) during the course of tandem mass spectrometry of deprotonated N-(phenylsulfonyl)-benzeneacetamide. Upon collisional activation, the formation of the initial ion/neutral complex ([C6H5CH2(-)/C6H5SO2NCO]), which was generated by heterolytic cleavage of the CH2-CO bond, is proposed as the key step. Subsequently, the anionic counterpart, benzyl anion, is transferred to conduct the intra-complex SNAr reaction. After losing neutral HNCO, the intermediate gives rise to product ion B at m/z 231, whose structure is confirmed by comparing the multistage spectra with those of deprotonated 2-benzylbenzenesulfinic acid and (benzylsulfonyl)benzene. In addition, intra-complex proton transfer is also observed within the complex [C6H5CH2(-)/C6H5SO2NCO] to generate product ion C at m/z 182. The INC-mediated mechanism was corroborated by theoretical calculations, isotope experiments, breakdown curve, substituent experiments, etc. This work may provide further understanding of the physicochemical properties of the gaseous benzyl anion.

A mechanistic study of fragmentation of deprotonated N,2-diphenyl-acetamides in electrospray ionization tandem mass spectrometry.

RATIONALE: Exploring the fragmentation mechanism of amide ions in mass spectrometry has attracted great interest because of the desire to analyze the amino acid sequences of peptides and proteins. However, the collision-induced dissociation (CID) mechanism of deprotonated small amides has been rarely studied in electrospray ionization mass spectrometry (ESI-MS). The fragmentation of deprotonated N,2-diphenylacetamides exhibited some characteristic fragment ions, which are not derived from the conventional cleavage route. Therefore, clarification of their fragmentation mechanism is very important and useful for structural analysis of related amides and peptides. METHODS: All CID experiments were carried out using an electrospray ionization ion trap mass spectrometer in negative ion mode. In addition, the accurate masses of fragments were measured on an ESI quadrupole time-of-flight (Q-TOF) mass spectrometer in negative ion mode. Deuterium-labeled 2-phenyl-N-(4-trifluoromethylphenyl)acetamide was synthesized and its ESI fragmentation spectrum had been obtained. Theoretical calculations were carried out by the density functional theory (DFT) method at the B3LYP level of theory with the 6-31G++(d,p) basis set. RESULTS: Deprotonated N,2-diphenylacetamides mainly generate four kinds of ions in CID: benzyl anion, aniline anion, phenyl-ethenone anion and isocyanato-benzene anion bearing respective substituent groups. The benzyl anion and the aniline anion can be generated by direct decomposition. The phenyl-ethenone anion and the isocyanato-benzene anion were proposed to be yielded from proton transfer within an ion-neutral complex, and the intensities of two competitive product ions are well correlated with the substituent constants. The mechanism was also supported by theoretical calculations. CONCLUSIONS: The characteristic fragment ions of deprotonated N,2-diphenylacetamides were proposed to be produced via an ion-neutral complex mechanism, which was proved by deuterium-labeling experiments, theoretical calculations and substituent effects.

Gas-Phase Alcoholysis of Benzylic Halides in the Atmospheric Pressure Ionization Source.

The present study investigates the gas-phase alcoholysis reaction of benzylic halides under atmospheric pressure chemical ionization (APCI) conditions. The APCI corona discharge is used to initiate the novel reaction, which is monitored by ion trap mass spectrometry (IT-MS). The model compound α,α,α-trifluorotoluene is applied to observe the cascade methoxylation reaction during the +APCI-MS analysis, resulting in the formation of [PhC(OCH3)2]+. Based on the results of isotopic labeling and substrate expansion experiments, an addition-elimination mechanism is proposed: initially, the reaction was initiated by the dissociation of fluorine from PhCF3 under APCI condition, leading to the formation of [PhCF2]+; subsequently, two methanol molecules nucleophilicly attack [PhCF2]+ stepwisely, accompanied by the elimination of HF, yielding the product ion [PhC(OCH3)2]+. The proposed mechanism was further corroborated by theoretical calculations. The results of substrate scope expansion experiments suggest that this in-source reaction has the potential to differentiate the positional isomers of alcohols and phenols.

Does deprotonated benzoic acid lose carbon monoxide in collision-induced dissociation?

Decarboxylation is known to be the major fragmentation pathway for the deprotonated carboxylic acids in collision-induced dissociation (CID). However, in the CID mass spectrum of deprotonated benzoic acid (m/z 121) recorded on a Q-orbitrap mass spectrometer, the dominant peak was found to be m/z 93 instead of the anticipated m/z 77. Based on theoretical calculations, 18 O-isotope labeling and MS3 experiments, we demonstrated that the fragmentation of benzoate anion begins with decarboxylation, but the initial phenide anion (m/z 77) can react with trace O2 in the mass analyzer to produce phenolate anion (m/z 93) and other oxygen-containing ions. Thus oxygen adducts should be considered when annotating the MS/MS spectra of benzoic acids.

Insight into pyrrolizidine alkaloids degradation and the chemical structures of their degradation products using ultra high performance liquid chromatography and Q-Exactive Orbitrap mass spectrometry.

Pyrrolizidine alkaloids (PAs), released into the environment by donor plants, are absorbed by crops or transported by animals, posing a global food safety concern. Photolysis is an effective way to eliminate harmful substances in the environment or food. Photolysis happens as PAs move among plants, environment and crops. In this study, we first investigated the photolysis and hydrolysis of 15 PAs and identified their degradation products via ultra-high performance liquid chromatography and Q-Exactive Orbitrap mass spectrometry. PAs were degraded under UV radiation but minimally affected by visible light from a xenon lamp, and solvent pH had little impact on their photolysis. PAs were stable in neutral and acidic solutions but degraded by 50% within 24 h in alkaline conditions. The degradation products of PAs were mainly PAs/PANOs isomers and some minor byproducts. Cytotoxicity and computational analysis revealed isomers had similar toxicity, with minor products being less toxic. This study is a precursor for revealing the potential PAs degradation dynamics in the environment and food products, providing a reference for systematic evaluations of potential health and ecological risks of their degradation products.

Uptake, accumulation, translocation and transformation of seneciphylline (Sp) and seneciphylline-N-oxide (SpNO) by Camellia sinensis L.

Pyrrolizidine alkaloids (PAs) and their N-oxide (PANOs), as emerging environmental pollutants and chemical hazards in food, have become the focus of global attention. PAs/PANOs enter crops from soil and reach edible parts, but knowledge about their uptake and transport behavior in crops is currently limited. In this study, we chose tea (Camellia sinensis L.) as a representative crop and Sp/SpNO as typical PAs/PANOs to analyze their root uptake and transport mechanism. Tea roots efficiently absorbed Sp/SpNO, utilizing both passive and active transmembrane pathways. Sp predominantly concentrated in roots and SpNO efficiently translocated to above-ground parts. The prevalence of SpNO in cell-soluble fractions facilitated its translocation from roots to stems and leaves. In soil experiment, tea plants exhibited weaker capabilities for the uptake and transport of Sp/SpNO compared to hydroponic conditions, likely due to the swift degradation of these compounds in the soil. Moreover, a noteworthy interconversion between Sp and SpNO in tea plants indicated a preference for reducing SpNO to Sp. These findings represent a significant stride in understanding the accumulation and movement mechanisms of Sp/SpNO in tea plants. The insights garnered from this study are pivotal for evaluating the associated risks of PAs/PANOs and formulating effective control strategies.

Identification and characterization of phenolamides in tea (Camellia sinensis) flowers using ultra-high-performance liquid chromatography/Q-Exactive orbitrap mass spectrometry.

Phenolamides (PAs) are important secondary metabolites present in plants with multiple bioactivities. This study aims to comprehensively identify and characterize PAs in tea (Camellia sinensis) flowers using ultra-high-performance liquid chromatography/Q-Exactive orbitrap mass spectrometry based on a lab-developed in-silico accurate-mass database. The PAs found in tea flowers were conjugates of Z/E-hydroxycinnamic acids (p-coumaric, caffeic and ferulic acids) with polyamines (putrescine, spermidine and agmatine). The positional and Z/E isomers were distinguished through characteristic MS2 fragmentation rules and chromatographic retention behavior summarized from some synthetic PAs. 21 types of PAs consisting of over 80 isomers were identified, and the majority of them were found in tea flowers for the first time. Among 12 tea flower varieties studied, they all possessed tris-(p-coumaroyl)-spermidine with the highest relative content, and C. sinensis 'Huangjinya' had the highest total relative contents of PAs. This study shows the richness and structural diversity of PAs in tea flowers.

Near-infrared-excitable acetylcholinesterase-activated fluorescent probe for sensitive and anti-interference detection of pesticides in colored food.

The development of a common and anti-interference acetylcholinesterase (AChE) inhibition assay for plant-originated food samples has been of great challenge because of the prevalent and strong signal interferences from natural pigments. Plant pigments normally exhibit non-negligible absorbance in the UV-visible region. As a result, the signals of a typical near-infrared (NIR) fluorescent probe could be disturbed through primary inner filter effect if it is excited by UV-visible light during plant sample analysis. In this work, an NIR-excitable AChE-activated fluorescent probe was biomimetically designed and synthesized. And the NIR-excitation strategy was utilized for the anti-interference detection of organophosphate and carbamate pesticides in colored samples with this probe. Sensitive and rapid response to AChE and pesticides was achieved due to the high affinity of the biomimetic recognition unit in the probe. The limits of detection for four representative pesticides including dichlorvos, carbofuran, chlorpyrifos and methamidophos reached 0.0186 µg/L, 2.20 µg/L, 12.3 µg/L and 13.6 µg/L, respectively. Most importantly, fluorescent response to pesticide contents could be accurately measured in the coexistence of different plant pigments by this probe, and the measured results showed completely irrelevance to the plant pigments and their colors. Taking advantage of such probe, the new developed AChE inhibition assay showed good sensitivity and anti-interference ability in the detection of organophosphate and carbamate pesticides in real samples.

Uptake and translocation of organic pollutants in Camellia sinensis (L.): a review.

Camellia sinensis (L.) is a perennial evergreen woody plant with the potential for environmental pollution due to its unique growth environment and extended growth cycle. Pollution sources and pathways for tea plants encompass various factors, including atmospheric deposition, agricultural inputs of chemical fertilizers and pesticide, uptake from soil, and sewage irrigation. During the cultivation phase, Camellia sinensis (L.) can absorb organic pollutants through its roots and leaves. This review provides an overview of the uptake and translocation mechanisms involving the absorption of polycyclic aromatic hydrocarbons (PAHs), pesticides, anthraquinone (AQ), perchlorate, and other organic pollutants by tea plant roots. Additionally, we summarize how fresh tea leaves can be impacted by spraying pesticide and atmospheric sedimentation. In conclusion, this review highlights current research progress in understanding the pollution risks associated with Camellia sinensis (L.) and its products, emphasizing the need for further investigation and providing insights into potential future directions for research in this field.

Dissociative benzyl cation transfer versus proton transfer: loss of benzene from protonated N-benzylaniline.

In collisional activation of protonated N-benzylaniline, the benzene loss from the benzyl moiety is actually not the result of dissociative proton transfer (PT). In fact, benzyl cation transfer (BCT) from the nitrogen to the anilinic ring (ortho or para position) is the key step for benzene loss. Such dissociation occurs only after the benzyl group migrating from the site with the highest benzylation nucleophilicity (nitrogen) to a different one (aromatic ring carbon), which is described as dissociative benzyl cation transfer.

Tandem mass spectrometric analysis and density functional theory calculations on the fragmentation behavior of two tetradecanoylingenol regioisomers from Euphorbia wallichii.

RATIONALE: Two structurally similar bioactive regioisomers, 3-O-tetradecanoylingenol and 20-O-tetradecanoylingenol, from Euphorbia wallichii presented quite different fragmentation behaviors. Revealing the related fragmentation pathways may improve the efficiency of characterization and identification of such type of compounds. METHODS: The two regioisomers were subjected to collision-induced dissociation (CID) on Finnigan LCQ(DECA) and LTQ Orbitrap XL instruments. Based on the CID results, the possible fragmentation pathways were proposed and investigated with density functional theory (DFT) calculations. RESULTS: Elimination of C(14)H(28)O(2) (tetradecanoic acid) for 3-O-tetradecanoylingenol and the sequential losses of C(4)H(8) (butylene) for 20-O-tetradecanoylingenol were observed in ESI-MS/MS, witnessed also by HR-ESI-MS/MS. The fragmentation pathways were proposed and verified by calculating the activation energy of their transition states by DFT calculations. CONCLUSIONS: Based on the observations, fragmentation pathways for the two regioisomers were proposed and verified by calculating the energy of the reactants, products and the corresponding transition states using DFT. This report should have value in rapid identification of the derivatives of ingenol and other regioisomers in natural products.

The antioxidant effect of imine resveratrol analogues.

Twenty five Imine resveratrol analogues (IRAs) were synthesized, replacing the C=C bond in resveratrol with CN bond, as well as substitution modifications on aromatic rings. Radical scavenging activities against DPPH, along with singlet oxygen quenching capacities were evaluated, and further confirmed using density functional theory calculations (DFT). It was found that IRAs bearing ortho-OH on B ring have better radical scavenging activities against DPPH than resveratrol, these compounds were also found to be effective (1)O(2) quenchers. Theoretical studies on the reaction mechanism of these compounds with (1)O(2) suggest that the 1,3-addition to a double bond with a -OH group with the formation of allylic hydroperoxide is the most probable reaction route.

Cα-Cß and Cα-N bond cleavage in the dissociation of protonated N-benzyllactams: dissociative proton transfer and intramolecular proton-transport catalysis.

In mass spectrometry of protonated N-benzylbutyrolactams, the added proton is initially localized on the carbonyl oxygen, which is the thermodynamically preferred protonation site. Upon collisional activation, dissociative proton transfer takes place leading to the occurrence of fragmentation reactions. The major fragmentations observed are the cleavages of C(α)-C(ß) and C(α)-N bonds on the two sides of the methylene linker, which is different to the cleavage of the amide bond itself seen in most amide cases. Theoretical calculations and isotopic labeling experiments demonstrate that the phenyl ring regulates the proton transfer reactions. The proton directly migrates to the C(ß) position via a 1,5-H shift leading to the efficient loss of benzene, while it stepwise migrates to the amide nitrogen resulting in the formation of a benzyl cation. The stepwise proton transfer is achieved via intramolecular proton-transport catalysis. The C(γ) position accepts the proton from the carbonyl oxygen via a 1,6-H shift, and then donates it to the amide nitrogen via a 1,4-H shift. The general 1,3-H shift from the carbonyl oxygen to the amide nitrogen can be excluded in this case due to its significant energy barrier. The substituent effects are also applied to explore the reaction mechanism, and it proves that both C(ß) and C(γ) are involved in the dissociative proton transfer processes. For monosubstituted N-benzylbutyrolactams, the abundance ratios of the two competing product ions are well correlated with the nature of the substituents.