Urinary phenylethylamine metabolites as potential markers for sports drug testing purposes

The misuse of 2-phenylethylamine (PEA) in sporting competitions is prohibited by the World Anti-Doping Agency. As it is endogenously produced, a method is required to differentiate between naturally elevated levels of PEA and the illicit administration of the drug. In 2015, a sulfo-conjugated metabolite [2-(2-hydroxyphenyl)acetamide sulfate (M1)] was identified, and pilot study data suggested that the ratio M1/PEA could be used as a marker indicating the oral application of PEA. Within this project, the required reference material of M1 was synthesized, single and multiple dose elimination studies were conducted and 369 native urine samples of athletes were analyzed as a reference population. While the oral administration of only 100 mg PEA did not affect urinary PEA concentrations, an increase in urinary concentrations of M1 was observed for all volunteers. However, urinary concentrations of both PEA and M1 showed relatively large inter-individual differences and establishing a cut-off-level for M1/PEA proved difficult. Consequently, a second metabolite, phenylacetylglutamine, was considered. Binary logistic regression demonstrated a significant (P < 0.05) correlation of the urinary M1 and phenylacetylglutamine concentrations with an oral administration of PEA, suggesting that assessing both analytes can assist doping control laboratories in identifying PEA misuse.     

Comparison of in‐source collision‐induced dissociation and beam‐type collision‐induced dissociation of emerging synthetic drugs using a high‐resolution quadrupole time‐of‐flight mass spectrometer

In‐source collision‐induced dissociation (CID) is commonly used with single‐stage high‐resolution mass spectrometers to gather both a molecular formula and structural information through the collisional activation of analytes with residual background gas in the source region of the mass spectrometer. However, unlike tandem mass spectrometry, in‐source CID does not involve an isolation step prior to collisional activation leading to a product ion spectrum composed of fragment ions from any analyte present during the activation event. This work provides the first comparison of in‐source CID and beam‐type CID spectra of emerging synthetic drugs on the same instrument to understand the fragmentation differences between the two techniques and to contribute to the scientific foundations of in‐source CID. Electrospray ionization–quadrupole time‐of‐flight (ESI‐Q‐TOF) mass spectrometry was used to generate product ion spectra from in‐source CID and beam‐type CID for a series of well‐characterized fentanyl analogs and synthetic cathinones. A comparison between the fragmentation patterns and relative ion abundances for each technique was performed over a range of fragmentor offset voltages for in‐source CID and a range of collision energies for beam‐type CID. The results indicate that large fragmentor potentials for in‐source CID tend to favor higher energy fragmentation pathways that result in both kinetically favored pathways and consecutive neutral losses, both of which produce more abundant lower mass product ions relative to beam‐type CID. Although conditions can be found in which in‐source CID and beam‐type CID provide similar overall spectra, the in‐source CID spectra tend to contain elevated noise and additional chemical background peaks relative to beam‐type CID.     

Gas‐phase formation of protonated benzene during collision‐induced dissociation of certain protonated mono‐substituted aromatic molecules produced in electrospray ionization

Protonated benzene, C₆H, has been studied extensively to understand the structure and energy of a protonated organic molecule in the gas phase. The formation of C₆H is either through direct protonation of benzene, i.e., chemical ionization, or through fragmentation of certain radical cations produced from electron ionization or photon ionization. We report a novel observation of C₆H as a product ion formed in the collision‐induced dissociation (CID) of protonated benzamide and related molecules produced via electrospray ionization (ESI). The formation of C₆H from these even‐electron precursor ions during the CID process, which has not been previously reported, is proposed to occur from the protonated molecules via a proton migration in a five‐membered ring intermediate followed by the cleavage of the mono‐substituent C? C bond and concurrent formation of an ion‐molecule complex. This unique mechanism has been scrutinized by examining some deuterated molecules and a series of structurally related model compounds. This finding provides a convenient mean to generate C₆H, a reactive intermediate of considerable interest, for further physical or chemical investigation. Further studies indicate that the occurrence of C₆H in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) appears to be a rather common phenomenon for many compounds that contain ‘benzoyl‐type’ moieties. Hence, the observation of the C₆H ion in LC/ESI‐MS/MS can be used as an informative fragmentation pathway which should facilitate the identification of a great number of compounds containing the ‘benzoyl‐type’ and similar structural features. These compounds are frequently present in food and pharmaceutical products as leachable impurities that require strict control and rapid elucidation of their identities. Copyright © 2010 John Wiley & Sons, Ltd.     

Energetic switch of the proline effect in collision‐induced dissociation of singly and doubly protonated peptide Ala‐Ala‐Arg‐Pro‐Ala‐Ala

Suppression of the selective cleavage at N‐terminal of proline is observed in the peptide cleavage by proteolytic enzyme trypsin and in the fragment ion mass spectra of peptides containing Arg‐Pro sequence. An insight into the fragmentation mechanism of the influence of arginine residue on the proline effect can help in prediction of mass spectra and in protein structure analysis. In this work, collision‐induced dissociation spectra of singly and doubly charged peptide AARPAA were studied by ESI MS/MS and theoretical calculation methods. The proline effect was evaluated by comparing the experimental ratio of fragments originated from cleavage of different amide bonds. The results revealed that the backbone amide bond cleavage was selected by the energy barrier height of the fragmentation pathway although the strong proton affinity of the Arg side chain affected the stereostructure of the peptide and the dissociation mechanism. The thermodynamic stability of the fragment ions played a secondary role in the abundance ratio of fragments generated via different pathways. Fragmentation studies of protonated peptide AACitPAA supported the energy‐dependent hypothesis. The results provide an explanation to the long‐term arguments between the steric conflict and the proton mobility mechanisms of proline effect.     

Heterocyclic ring cleavage upon collision‐induced dissociation of deprotonated 3‐hydroxy‐1,2,5‐oxadiazoles (3‐hydroxyfurazans)

A series of 4‐substituted 3‐hydroxyfurazans were subjected to electrospray ionization tandem mass spectrometry. At low collision energy, oxyisocyanate ([O=C=N–O]^?, m/z 58) was formed as the predominant product ion from each deprotonated 3‐hydroxyfurazan, indicating cleavage of the heterocyclic ring. The facile energetics of this characteristic fragmentation process was confirmed by density functional computations.     

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

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

Characterization of amino acid‐derived betaines by electrospray ionization tandem mass spectrometry

Betaines belong to the naturally occurring osmoprotectants or compatible solutes present in a variety of plants, animals and microorganisms. In recent years, metabolomic techniques have been emerging as a fundamental tool for biologists because the constellation of these molecules and their relative proportions provide with information about the actual biochemical condition of a biological system. Therefore, identification and characterization of biologically important betaines are crucial, especially for metabolomic studies. Most of the natural betaines are derived from amino acids and related homologues. Although, theoretically, all the amino acids can be converted to corresponding betaines by simple methylation of the amine group, only a few of the amino acid‐derived betaines were fully characterized in the literature. Here, we report a combined electrospray ionization tandem and high‐resolution mass spectrometry study of all the betaines derived from amino acids, including the isomeric betaines. The decomposition pathway of protonated, sodiated and potassiated molecule ions that enable unambiguous characterization of the betaines including the isomeric betaines and overlapping ionic species of different betaines is distinctive. Copyright © 2012 John Wiley & Sons, Ltd.     

VUV photon‐induced ionization/dissociation of antipyrine and propyphenazone: mass spectrometric and theoretical insights

Two analgesic and anti‐inflammatory drugs, antipyrine and propyphenazone, were investigated with infrared laser desorption/tunable synchrotron vacuum ultraviolet (VUV) photoionization mass spectrometry (IR LD/VUV PIMS) and theoretical calculations. Mass spectra of the two drugs were measured at various photon energies. Fragment ions were gradually produced as photon energy increases. The structural assignment of the dominant fragment ions was supported by the results from a commercial electron impact time‐of‐flight mass spectrometer (EI‐TOF MS). Primary fragmentation pathways were established from experimental observations combining with theoretical calculations. Methyl radical elimination is a common fragmentation pathway for two analytes. However, for propyphenazone cation, isopropyl group elimination to form antipyrine cation is another competitive pathway. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of multiple fragmentation pathways initiated by collision‐induced dissociation of multifunctional anions formed by deprotonation of 2‐nitrobenzenesulfonylglycine

The correlation of anion structure with the fragmentation behavior of deprotonated nitrobenzenesulfonylamino acids was investigated using tandem mass spectrometry, isotopic labeling and computational methods. Four distinct fragmentation pathways resulting from the collision‐induced dissociation (CID) of deprotonated 2‐nitrobenzenesulfonylglycine (NsGly) were characterized. The unusual loss of the aryl nitro substituent as HONO was the lowest energy process. Subsequent successive losses of CO, HCN and SO₂ indicated that an ortho cyclization reaction had accompanied loss of HONO. Other pathways involving rearrangement of the ionized sulfonamide group, dual bond cleavage and intramolecular nucleophilic displacement were proposed to account for the formation of phenoxide, arylsulfinate and arylsulfonamide product ions at higher collision energies. The four distinct fragmentation pathways were consistent with precursor–product relationships established by CID experiments, isotopic labeling results and the formation of analogous product ions from 2,4‐dinitrobenzenesulfonylglycine and the Ns derivatives of alanine and 2‐aminoisobutyric acid. The computations confirmed a low barrier for ortho cyclization with loss of HONO and feasible energetics for each reaction step in the four pathways. Computations also indicated that three of the fragmentation pathways started from NsGly ionized at the carboxyl group. Overall, the pathways identified for the fragmentation of the NsGly anion differed from processes reported for anions containing a single functional group, demonstrating the importance of functional group interactions in the fragmentation pathways of multifunctional anions. Copyright © 2014 John Wiley & Sons, Ltd.     

Accurate identification of dimers from α‐pinene oxidation using high‐resolution collision‐induced dissociation mass spectrometry

Interest in mass spectrometry of highly oxidized dimers from α‐pinene oxidation has increased in the atmospheric chemistry field. Here, we apply high‐resolution collision‐induced dissociation mass spectrometry (HR‐CID‐MS) with an atmospheric pressure ionization source to investigate in detail how α‐pinene‐derived dimers are detected and identified by MS. The resulting HR‐CID spectra and specific fragmentation patterns suggest that a large fraction of dimer ions detected in full‐scan mass spectra can be hydrogen‐bonded artifact clusters and the residual small fraction includes covalently bonded actual dimers. We also show how individual fractions of the artifact clusters and actual dimers are calculated using the HR‐CID spectra.     

Fingerprinting the degradation product patterns of different polyester‐ether networks by electrospray ionization mass spectrometry

Fingerprinting of the degradation product patterns by electrospray ionization mass spectrometry (ESI‐MS) was evaluated as a tool to monitor the degree of degradation in polyester‐ether networks. Four different crosslinked caprolactone (CL) and/or 1,5‐dioxepan‐2‐one (DXO) networks were subjected to hydrolytic degradation in aqueous solution at 37 °C for up to 147 days. After predetermined time periods, the water‐soluble degradation products were analyzed by ESI‐MS and tandem ESI‐MS. In addition, changes in pH, mass loss, and copolymer composition were determined. In the case of more slowly hydrolyzed CL‐rich (co)polymers, CL and/or DXO oligomers terminated by hydroxyl and carboxyl end groups were predominantly formed as degradation products. However, on prolonged hydrolysis oligomers with attached crosslinking agent dominated the degradation product patterns of more easily hydrolyzed DXO‐rich (co)polymers. It was shown that in the recorded mass spectra the variation of intensities in the series of ions corresponding to DXO and CL/DXO oligomers with or without attached crosslinking agent could be utilized to monitor the extent of hydrolytic degradation in the polyester matrix and the disruption of the network structure. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 4617–4629, 2008     

Energy‐dependent normal and unusually large inverse chlorine kinetic isotope effects of simple chlorohydrocarbons in collision‐induced dissociation by gas chromatography‐electron ionization‐tandem mass spectrometry

Kinetic isotope effects (KIEs) occurring in mass spectrometry (MS) can provide in‐depth insights into the fragmentation behaviors of compounds of interest in MS. Yet, the fundamentals of KIEs in collision‐induced dissociation (CID) in tandem mass spectrometry (MS/MS) are unclear, and information about chlorine KIEs (Cl‐KIEs) of organochlorines in MS is particularly scarce. This study investigated the Cl‐KIEs of dichloromethane, trichloroethylene, and tetrachloroethylene during CID using gas chromatography‐electron ionization triple‐quadrupole MS/MS. Cl‐KIEs were evaluated with MS signal intensities. All the organochlorines presented large inverse Cl‐KIEs (<1, the departures of Cl‐KIEs from 1 denote the magnitudes of Cl‐KIEs), showing the largest magnitudes of 0.797, 0.910, and 0.892 at the highest collision energy (60 eV) for dichloromethane, trichloroethylene, and tetrachloroethylene, respectively. For dichloromethane, both intra‐ion and inter‐ion Cl‐KIEs were studied, within the ranges of 0.820–1.020 and 0.797–1.016, respectively, showing both normal and inverse Cl‐KIEs depending on collision energies. The observed Cl‐KIEs generally declined from large normal to extremely large inverse values with increasing collision energies from 0 to 60 eV but were inferred to be independent of MS signal intensities. The Cl‐KIEs are dominated by critical energies at low internal energies of precursor ions, resulting in normal Cl‐KIEs; while at high internal energies, the Cl‐KIEs are controlled by rotational barriers (or looseness/tightness of transition states), which lead to isotope‐competitive reactions in dechlorination and thereby inverse Cl‐KIEs. It is concluded that the Cl‐KIEs may depend on critical energies, bond strengths, available internal energies, and transition state looseness/tightness. The findings of this study yield new insights into the fundamentals of Cl‐KIEs of organochlorines during CID and may be conducive to elucidating the underlying mechanisms of KIEs in collision‐induced and photo‐induced reactions in the actual world.     

Study of active naphtodianthrone St John's Wort compounds by electrospray ionization Fourier transform ion cyclotron resonance and multi‐stage mass spectrometry in sustained off‐resonance irradiation collision‐induced dissociation and infrared multiphoton dissociation modes

Five well‐known active naphtodianthrone constituents of Hypericum perforatum (St John's Wort) extracts have been investigated by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI‐FTICRMS) and ESI‐FTICRMSⁿ. The studied compounds were hypericin, pseudohypericin, protohypericin, protopseudohypericin (biosynthetic precursors of the two former compounds, respectively) and isopseudohypericin (alkaline degradation product of pseudohypericin). Dissociation mass spectrometry measurements performed on the [M–H]^? ion presented a variable efficiency as a function of the used activation mode. Sustained off‐resonance irradiation collision‐induced dissociation (SORI–CID) only led to a restricted number of fragment ions. In contrast, IRMPD ensured the detection of numerous product ions. Ions detected in ESI‐FTICRMS and ESI‐FTICRMSⁿ experiments were measured with a very high mass accuracy (typically mass error is lower than 0.5 mDa at m/z close to 500) that allowed unambiguous formulae to be assigned to each signal observed in a mass spectrum. In spite of similar structures, specific fragmentation patterns were observed for the different compounds investigated. This study may be useful in the future to characterize in natural extracts these compounds (or derivatives of these compounds) by liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments by considering the MS/MS transitions highlighted in this paper. Copyright © 2009 John Wiley & Sons, Ltd.     

Degradation of poly(butyl methacrylate) model compounds studied via high‐resolution electrospray ionization mass spectrometry

This study investigates the degradation behavior of poly(n‐butyl methacrylate) ( p(nBMA) ), poly(tert‐butyl methacrylate) ( p(tBMA) ), and poly(hexafluoro butyl methacrylate) ( p(HFBMA) ) on a molecular level under extreme environmental conditions. The polymers chosen are readily applicable in the formulation of surface coatings and were degraded under conditions which replicated the harsh Australian climate, in which surface coatings may reach temperatures of up to 95 °C and are exposed to broad‐spectrum UV radiation of up to 1 kW m^?2. The degradation profiles were mapped with high‐resolution electrospray ionization mass spectrometry (ESI‐MS) with a LCQ quadrupole ion trap mass analyzer, with the peak assignments confirmed to within 3 ppm using ESI‐MS with a LTQ‐Orbitrap mass detector. It was found that in all the butyl ester polymers analyzed herein—regardless of their tertiary side‐chain structure—the loss of the butyl ester group and subsequent formation of acid side groups are a component of the overall degradation pathway of poly(butyl methacrylate)s under these harsh conditions. However, it is also demonstrated that the magnitude of this pathway is intimately linked to the side‐chain structure with the propensity for degradation decreasing in the order p(tBMA) > p(nBMA) > p(HFBMA) . The degradation mechanisms identified in this study, in combination with the previous end‐group degradation studies of poly(methyl methacrylate) and poly(n‐butyl acrylate), have allowed a much deeper understanding of the molecular degradation behavior of poly(acrylate)s and poly(methacrylate)s in an extreme natural environment. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011