In electrospray ionization source hydrogen/deuterium exchange LC-MS and LC-MS/MS for characterization of metabolites

A new method is described for performing hydrogen/deuterium (H/D) exchange in an electrospray ionization (ESI) source. The use of liquid chromatography (LC)-mass spectrometer equipped with an ESI source and deuterium oxide (D2O) as the sheath liquid allows H/D exchange experiments to be performed on-line. This directly provides information for determining the number and position of exchangeable hydrogens, aiding in the elucidation of the structures of drug metabolites. To demonstrate the utility of this method, LC-mass spectrometry (MS) and LC-MS/MS experiments were performed using either H2O or D2O as sheath liquid on a matrix metalloprotease (MMP) inhibitor (PD 0200126) and its metabolites. Examination of the mass shift of the deuteriated molecule from that of the protonated molecule allowed the number of exchangeable protons to be determined. Interpretation of the production-spectra helped to determine the location of the exchanged protons and assisted in the assignment of the site(s) of modification for each metabolite.     

Hydrogen/deuterium exchange using a coaxial sheath-flow interface for capillary electrophoresis/mass spectrometry

The interfacing of capillary electrophoresis (CE) with mass spectrometry (MS) is well established and may be accomplished by use of either a coaxial arrangement or by employing a liquid T-junction. In both these interfaces a make-up flow is introduced. This is required because of the mismatch in flow rates for capillary electrophoresis approximately nL/min and 'true' electrospray approximately 2-10 microL/min. Electrical connectivity may also be established where the liquid flows meet (the introduction of nanospray renders the use of make-up flow unnecessary). Hydrogen/deuterium (H/D) exchange occurs in solution when there are labile hydrogen atoms present in a molecule. The establishment of the presence and the number of such exchangeable hydrogen atoms may be of importance in the identification and differentiation of compounds. It may also be an aid in the structural elucidation of unknown materials. We have investigated the feasibility of carrying out H/D exchange via a CE/MS interface. This involved the addition of D2O to the sheath flow and our preliminary results showing the separations of drug substances, subsequently undergoing exchange, are presented.     

Methyl guanine isomer distinction by hydrogen / deuterium exchange using a fourier transform mass spectrometer

Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA Differentiation of the seven isomers of methyl guanine has been accomplished by monitoring gas-phase hydrogen/deuterium (H/D) exchange reactions of the protonated molecular ions with deuterium oxide (D₂O) in a Fourier transform mass spectrometer. In each case a distinctive reaction rate for the first H/D exchange was observed, and exchanges of up to three deuterium atoms occurred with characteristic ion abundances that could be used to differentiate the isomers. O⁶-Methyl guanine, for example, showed only one slow H/D exchange with D₂O, whereas l-methyl guanine exchanged two hydrogen atoms at a significantly faster rate. On comparison of the possible resonance structures of each protonated isomer with the experimental information about the number and rate of H/D exchanges observed, a reaction mechanism involving a concerted proton abstraction-deuterium cation donation was proposed.     

Low‐Temperature Alkane C?H Bond Activation by Zeolites: An In Situ Solid‐State NMR H/D Exchange Study for a Carbenium Concerto

Isotopic H/D exchange has been monitored by in situ MAS NMR spectroscopy of 2‐[D₁₄]methylpentane with H‐USY to probe the controversy over the alkane conversion mechanism. The probe molecule has distinct exchangeable sites with different accessibility to the zeolite surface. In the early stages of the process, the regioselectivity of exchange demonstrates that the slow step of the mechanism is controlled by a carbenium ion intermediate. At a later stage of exchange, intramolecular hydride migrations, typical of carbenium chemistry, replace D by H also on other carbon atoms, resulting in a loss of regioselectivity. Therefore, the first and the subsequent steps of the H/D exchange proceed at this temperature through a carbenium intermediate species.     

Hydrogen/deuterium exchange for higher specificity of protein identification by peptide mass fingerprinting

Genome sequencing projects produce large amounts of information that could be translated into potential protein sequences. Such amounts of material continuously increase protein database sizes. At present, 22 times more protein sequences are available in the SWISS-PROT and TrEMBL databases than 8 years ago in SWISS-PROT. One of the methods of choice for protein identification makes use of specific endoproteolytic cleavage followed by matrix-assisted laser desorption/ionisation mass spectrometric (MALDI-MS) analysis of the digested product. Since 1993, when this technique was first demonstrated, the conditions required for a correct identification have changed dramatically. Whilst 4-5 peptides with an uncertainty of 2-3 Da were sufficient for a correct identification in 1993, 10-13 peptides with less than 60 ppm mass error are now required for human and E. coli proteins. This evolution is directly related to the continuous increase in protein database sizes, which causes an increase in the number of false positive matches in identification results. Use of an information complement deduced from the primary protein sequence, in the process of identification by peptide mass fingerprints, can help to increase confidence in the identification results. In this article, we propose the exchange of labile hydrogen atoms with deuterium atoms to provide an alternative information complement. The exchange reaction with optimised techniques has shown an average 95% of hydrogen/deuterium (H/D) exchange on tryptic peptides. This level of exchange was sufficient to single out one or more peptides from a list of potential candidate proteins due to the dependence of H/D exchange on the peptide primary structure. This technique also has clear advantages in the identification of small proteins where direct protein identification is impaired by the limited number of endoproteolytic peptides. Then, information related to primary sequence obtained with this technique could help to identify proteins with high confidence without any expensive tandem mass spectrometry instruments.     

Reactive collisions in quadrupole cells. 2. H/D exchange reactions of enolate ions with CH3od and C2H5OD

The H/D exchange reactions of enolate ions with CH₃OD and C₂H₅OD at low collision energies in the quadrupole collision cell of a hybrid tandem BEqQ mass spectrometer have been studied. The optimum parameters for exchange were elucidated using the enolate ion of acetone as a test reactant ion. With the exception of the enolate ions of 2,4-pentanedione and dimethylmalonate, all the enolate ions studied underwent exchange with CH₃OD and C₂H₅OD. Ion signals were observed corresponding to exchange of from one to all of the enolic hydrogen atoms. The H/D exchange reaction was more facile with C₂H₅OD as the reagent gas, and the results with this gas clearly permit a count of the number of enolic hydrogen atoms present in the molecule.     

Selecting high stability water clusters by examining locations of free OH bonds: Application in the study of clusters

Computational studies on water clusters can be quite challenging, especially when an irregular cage with non-equivalent oxygen sites are considered which may yield a large number of starting geometries that differ in relative positions of non-H-bonding H (NHB H, free OH) atoms. A detailed study on water octamers suggests that the fewest number of NHB H atoms on neighboring oxygen sites yields the most stable neutral isomer followed by those with increasing number of NHB H atoms on adjacent sites. The least stable cluster has all the NHB H atoms around a ring. By considering a regular cage structure and minimum number of NHB H atoms on adjacent sites, one can readily identify a limited number of starting geometries that are optimized to highly stable isomers. This method has been verified in the identification of the most stable isomer of (H₂O)₈ cubic cage and (H₂O)₂₀ dodecahedral cage. The same method has been applied in the study of cluster isomers.     

Determination of exchangeable protons in natural organic matter using a home-made hydrogen/carbon analyser

A home made hydrogen/carbon analyser was used to determine the portion of exchangeable protons in aquatic humic substances. For this purpose, equal sample amounts were dissolved in H2O and D2O, respectively, dried and combusted in a stream of oxygen. The amount of water resulting from combustion was measured by an infrared detector which recorded the OH bending vibration of H2O. The bands stemming from HOD or D2O were not registered by the detection unit. Thus, combustion of organic samples containing exchangeable protons dissolved in D2O resulted in a significantly smaller signal compared to the signal observed for the same sample dissolved in H2O. The relative intensity loss of the H2O signal observed after combustion was used to derive the portion of exchangeable protons in a standard reference material, a humic substance isolated by the International Humic Substances Society (Suwannee River fulvic acid). According to this method about 20% of the sample protons could be identified as exchangeable protons. With regard to titration data the portion of protons bound to non acidic hydroxy functions could be estimated. The validity of this procedure was proved by combustion experiments using commercially available deuterated substances as well as organic model compounds dissolved in D2O and H2O, respectively.     

Kinetics of gas-phase hydrogen/deuterium exchange and gas-phase structure of protonated phenylalanine, proline, tyrosine and tryptophan

Site-specific rate constants for the gas-phase hydrogen/deuterium (H/D) exchange of four, three, five and five hydrogen atoms in protonated phenylalanine (Phe), proline (Pro), tyrosine (Tyr) and tryptophan (Trp), respectively, were determined from matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICRMS) experiments with D(2)O, D(2)S, and CH(3)OD as deuterating agents. No H/D exchange was observed with D(2)S. For exchange with both CD(3)OD and D(2)O, which is about ten times slower in the latter, results indicate for all compounds protonation of the alpha-amino group in agreement with theoretical results. Also, with both reagents, all compounds exchange at the COOH site more than ten times faster than at the protonation site, with OH and NH sites of Tyr and Trp, respectively, exchanging slowest. The observation of H/D exchange despite the high differences in proton affinities between the amino acids and deuterating agent exceeding 200 kJ mol(-1) is in agreement with lowering of the barrier for proton transfer through hydrogen bonding proposed by Lebrilla and coworkers.     

A fast flow tube study of gas phase H/D exchange of multiply protonated ubiquitin

An electrospray ionization (ESI)/fast-flow technique has been applied to the study of gas phase hydrogen/deuterium (H/D) exchange kinetics. Multiply charged ubiquitin ions [ubiquitin + nH](n)(+), in charge states n = 7-13, were reacted with ND(3). The behavior of ND(3) as exchange reagent is different from that of the previously studied reagents, D(2)O and CH(3)OD. Contrary to those, the maximum number of exchanged hydrogen atoms and the overall exchange rate were observed to increase with increasing charge state of the ubiquitin ions. The results are reagent-dependent because the exchange mechanisms are different for the different reagents. This observation is in agreement with a recent conclusion by Beauchamp and co-workers that contrary to the assumption often expressed in earlier studies, H/D exchange kinetics may not directly reflect ion structures. The results for all three reagents are, however, consistent with observations of previous ion mobility experiments that with increasing charge state the conformers change from more compact, partially folded structures to elongated nearly linear ones. H/D exchange of (ubiquitin + 13H)(13+) with ND(3) leads to two separated ion populations reflecting the possible existence of two conformers with different exchange rates. The ions (ubiquitin + 8H)(8+) and (ubiquitin + 11H)(11+) represent a partially folded structure and an unfolded structure, respectively, and were studied in greater detail. The relative abundances of ions were measured in steps of 0.5 m/z (mass-to-charge ratio), as a function of the ND(3) flow rate. The experimental results were simulated by computer fitted curves based on a recently developed algorithm. The algorithm allows the extraction of sets of grouped rate constants. Eight rate constant groups were deduced for each of the two ions. These rate constants correspond to 32 and 44 H/D exchanges for the 8+ and 11+ charged ions, respectively. The results indicate higher individual rates for most of the exchanged atoms in the 11+ ion compared to the 8+ ion.     

Simplified batch equilibration for D/H determination of non‐exchangeable hydrogen in solid organic material

Hydrogen isotopic analysis of organic materials has been widely applied in studies of paleoclimate, animal migration, forensics, food and flavor authentication, and the origin and diagenesis of organic matter. Hydrogen bound to carbon (C‐H) generally retains isotopic information about the water present during organic matter synthesis and associated biosynthetic fractionations, but hydrogen bound to other elements (O, S, or N) can readily exchange with atmospheric water vapor and reflects recent exposure to water or vapor. These two pools must be separated to obtain meaningful information from isotope ratios of organic materials. Previously published analytical methods either replace exchangeable H chemically or control its isotopic composition, usually by equilibration with water or waters of known isotopic composition. In addition, the fraction of H that is exchangeable can vary among samples and is itself of scientific interest. Here we report an improved and automated double‐equilibration approach. Samples are loaded in a 50‐position autosampler carousel in an air‐tight aluminum equilibration chamber. Water vapor of known isotopic composition is pumped through the chamber at 115°C for at least 6 h. After flushing with dry N₂ and being cooled, the carousel is rapidly transferred from the equilibration chamber to a He‐purged autosampler attached to a pyrolysis elemental analyzer connected to an isotope ratio mass spectrometer. By equilibrating two aliquots of each sample with two isotopically distinct waters, it is possible to calculate both (1) the D/H ratio of non‐exchangeable H, and (2) the fraction of H that is exchangeable. Relative to previous double‐equilibration techniques, this approach offers significant reductions in sample size and labor by allowing simultaneous equilibration of several tens of samples. Copyright © 2009 John Wiley & Sons, Ltd.     

环丙烷衍生物在D2O离子体系中的H/D交换反应

在D2O化学反应气条件下研究了环丙烷衍生物的H/D交换反应特性.发现了三种新的产物离子[M+1]+、[M+2]+和[M+3]+.应用碰撞诱导碎裂(CID)技术研究了这些离子的碎裂反应特性.实验结果表明三种新的产物离子是由反应物与试剂离子之间发生H/D交换反应生成的.并获得了环丙烷衍生物结构中活泼氢位置及其数量的信息.     

Conformational changes of ubiquitin during electrospray ionization as determined by in‐ESI source H/D exchange combined with high‐resolution MS and ECD fragmentation

In the paper, we have demonstrated the possibility of performing hydrogen/deuterium (H/D) exchange of proteins in the region of gas‐phase ion formation in an electrospray ion source by saturating the electrospray ionization source with vapors of a deuterating agent (D₂O or MeOD). In this region, charged droplets are shrinking and the protein ions transfer into the gas phase. As a model protein, we have used ubiquitin whose ion mobility spectrometry and gas‐phase H/D exchange in the vacuum part of a mass spectrometer demonstrated the presence of gas‐phase conformers with different cross sections and H/D exchange rates. In our experiments, we observed monomodal deuterium distributions for all solvents, charge states, desolvating capillary temperature and types of deuterating agent. Also, we found that the number of H/D exchanges increases with an increasing desolvating capillary temperature and decreasing charge state. We observed that solution composition (49 : 50 : 1 H₂O : MeOH : formic acid or 99 : 1 H₂O : formic acid) influences the charge‐state distribution but did not change the degree of H/D exchange for the same charge state. Electron‐capture dissociation fragmentation shows that higher charge states contain a segment that is protected from access by the deuterating agent. Copyright © 2014 John Wiley & Sons, Ltd.     

Computational investigation and hydrogen/deuterium exchange of the fixed charge derivative tris(2,4,6-Trimethoxyphenyl) phosphonium: Implications for the aspartic acid cleavage mechanism

Aspartic acid (Asp)-containing peptides with the fixed charge derivative tris(2,4,6trimethoxyphenyl) phosphonium (tTMP-P+) were explored computationally and experimentally by hydrogen/deuterium (H/D) exchange and by fragmentation studies to probe the phenomenon of selective cleavage C-terminal to Asp in the absence of a "mobile" proton. Ab initio modeling of the tTMP-P+ electrostatic potential shows that the positive charge is distributed on the phosphonium group and therefore is not initiating or directing fragmentation as would a "mobile" proton. Geometry optimizations and vibrational analyses of different Asp conformations show that the Asp structure with a hydrogen bond between the side-chain hydroxy and backbone carbonyl lies 2.8 kcal/mol above the lowest energy conformer. In reactions with D2O, the phosphonium-derived doubly charged peptide (H+)P+LDIFSDF rapidly exchanges all 12 of its exchangeable hydrogens for deuterium and also displays a nonexchanging population. With no added proton, P+LDIFSDF exchanges a maximum of 4 of 11 exchangeable hydrogens for deuterium. No exchange is observed when all acidic groups are converted to the corresponding methyl esters. Together, these H/D exchange results indicate that the acidic hydrogens are "mobile locally" because they are able to participate in exchange even in the absence of an added proton. Fragmentation of two distinct (H+)P+LDIFSDF ion populations shows that the nonexchanging population displays selective cleavage, whereas the exchanging population fragments more evenly across the peptide backbone. This result indicates that H/D exchange can sometimes distinguish between and provide a means of separation of different protonation motifs and that these protonation motifs can have an effect on the fragmentation.     

A study of the redox properties of MoOx/SiO2

A sample of MoOx/SiO2, in which all of the Mo cations are present as isolated mono-oxo molybdate moieties, was prepared and investigated to understand the redox chemistry of such molybdate species and their ability to exchange oxygen with O2 and H2O. Raman spectroscopy was used to monitor the exchange of 18O for 16O in the Mo=O bond of isolated molybdate species, whereas mass spectrometry was used to follow the isotopic composition of the gaseous species, i.e., O2 and H2O. Reduction in H2 at 920 K results in the loss of one O atom per Mo atom, and consistent with this, all of the Mo(VI) cations are reduced to Mo(IV) cations. Raman spectroscopy shows that virtually all Mo=O bonds of the original molybdate species are lost upon reduction. While reoxidation of Mo(IV) cations by O2 is quantitative, studies using 18O2 reveal that only a small part of the newly formed Mo=O bonds are 18O labeled, and that the balance are 16O labeled, indicating that O-atom exchange between the support, SiO2, and the supported MoOx species occurs during reoxidation. Rapid exchange of O atoms was observed upon exposure of both bare SiO2 and MoOx/SiO2 to H2(18)O at 920 K, and the presence of MoOx species was found to enhance the rate of exchange. By contrast, very slow exchange of O atoms was observed when the oxidized catalyst was exposed to 18O2 at 920 K. In situ observations of the catalyst during exposure to a mixture of H2 and 18O2 at 920 K showed that all of the Mo cations remained in the VI oxidation state and that O atom exchange occurred at a rate comparable to that observed upon exposure to H2(18)O. The results of this investigation suggest that reoxidation of Mo(IV) cations following H2 reduction involves the formation of a Mo-peroxide species and subsequent O atom migration from such a species to the SiO2 support. It is proposed that the steady-state oxidation of H2 also involves the formation of Mo-peroxide species by interaction of O2 with a small number of Mo(IV) centers. The Mo-peroxide species are then rapidly reduced by H2 to form H2O and a Mo=O bond. The rapid exchange of O atoms between the gas phase and the catalyst observed during steady-state oxidation of H2 is attributed to interactions of the product H2O with the catalyst, rather than to O atom migration originating from the Mo-peroxide species formed on the catalyst surface.     

Gas phase hydrogen / deuterium exchange in electrospray ionization mass spectrometry as a practical tool for structure elucidation

Two methods for gas phase hydrogen/deuterium exchange have been developed for the analysis of small molecules. Hydrogen/deuterium exchange has been implemented by making simple modifications to the plumbing for the nebulizer and curtain gases on a nebulization-assisted electrospray ion source. The nebulizer gas exchange method has demonstrated deuterium exchange levels of 84–97% for a variety of molecules representing a wide range of structural classes containing up to 51 potentially exchangeable hydrogens; this allowed determination of the number of exchangeable hydrogens for all of the molecules studied containing ≤ 25 labile hydrogens (M _r ≤ 3000). ND₃ gas consumption is minimized in the nebulizer method by toggling the nebulizer from air to ND₃ for only a few scans of the total sample elution period. The curtain gas exchange method is more variable, yielding exchange levels of 32–98% for the same set of molecules; this was still sufficient to allow determination of > 70% of the molecules studied containing ≤ 25 labile hydrogens. Gas consumption is minimized in the curtain method by replacing ≤ 10% of the curtain gas flow with ND₃. Neither the nebulizer nor curtain exchange method requires the use of deuterated or aprotic solvents at typical 2 μL/min flow rates.     

Hydrogen chemisorption on Al2O3-supported gold catalysts

Hydrogen is dissociatively adsorbed on the gold particles in Au/Al(2)O(3) catalysts, as demonstrated by a combination of in-situ X-ray absorption spectroscopy, chemisorption, and H/D exchange experiments. This chemisorption of hydrogen induces changes in the Au L(3) and L(2) X-ray absorption near-edge structures. The gold atoms on corner and edge positions dissociate the hydrogen, which does not spill over to the face sites. Therefore, the average number of adsorbed hydrogen atoms per surface gold atom increases with decreasing particle size. With temperature, the hydrogen uptake by supported gold increases or remains constant, whereas it decreases for platinum. Furthermore, in H/D exchange experiments, the activity of Au/Al(2)O(3) increases strongly with temperature. Thus, the dissociation and adsorption of hydrogen on gold is activated.     

In ESI‐source H/D exchange under atmospheric pressure for peptides and proteins of different molecular weights from 1 to 66 kDa: the role of the temperature of the desolvating capillary on H/D exchange

Transition of proteins from the solution to the gas phase during electrospray ionization remains a challenging problem despite the large amount of attention it has received during the past few decades. One of the major questions relates to the extent to which proteins in the gas phase retain their condensed phase structures. We have used in‐electrospray source hydrogen/deuterium exchange to determine the number of deuterium incorporations as a function of protein mass, charge state and temperature of the desolvating capillary where the reaction occurs. All experiments were performed on a Thermo LTQ FT Ultra equipped with a 7‐T superconducting magnet. Ions were generated by an IonMax Electrospray ion source operated in the positive ESI mode. Deuterium exchange was performed by introducing a droplet of D₂O beneath the ESI capillary. We systematically investigated gas phase hydrogen/deuterium (H/D) exchange under atmospheric pressure for peptides and proteins of different molecular weights from 1 to 66 kDa. We observed that almost all proteins demonstrate similar exchange rates for all charge states and that these rates increase exponentially with the temperature of the desolvating capillary. We did not observe any clear correlation of the number of H/D exchanges with the value of the cross section for a corresponding charge state. We have demonstrated the possibility of performing in‐ESI source H/D exchange of large proteins under atmospheric pressure. The simplicity of the experimental setup makes it a useful experimental technique that can be applied for the investigation of gas phase conformations of proteins. Copyright © 2015 John Wiley & Sons, Ltd.     

Liquid chromatography/quadrupole time‐of‐flight mass spectrometry in combination with online hydrogen/deuterium exchange technique for structural elucidation of phase I metabolites of iso‐phenylcyclopentylamine in rat bile

MS/MS experiment and accurate mass measurement are powerful tools in metabolite identification. However, sometimes these data do not provide enough information to assign an unambiguous structure to a metabolite. In combination with MS techniques, hydrogen/deuterium (H/D) exchange can provide additional information for structural elucidation by determination of the number of exchangeable hydrogen atoms in a structure. In this study, the principal phase I metabolites of iso‐phenylcyclopentylamine in rat bile were identified by high‐performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry (ESI‐Q‐TOF‐MS). Since N‐oxidation may occur because of the existence of the primary amino group in the structure, it was difficult to differentiate the hydroxylated metabolites from N‐oxides by ESI‐Q‐TOF‐MS alone. Therefore, online H/D exchange technique was applied to solve this problem. Finally, 25 phase I metabolites were detected and structurally described, in which 11 were confirmed to be N‐oxides. This study demonstrated the effectiveness of high‐resolution mass spectrometry in combination with an online H/D exchange technique in rapid identification of drug metabolites, especially in discriminating hydroxylated metabolites from N‐oxides. Copyright © 2014 John Wiley & Sons, Ltd.     

Isomer separation of hyperbranched polyesteramides with gas-phase H/D exchange and a novel MS(n) approach: DoDIP

Two approaches are introduced that provide information about the isomeric composition of hyperbranched polyesteramides. The first approach is based on a novel tandem mass spectrometric (MS(n)) approach that allows the study of different types of isomeric structures by a separation based on their difference in appearance energy. The method is called DoDIP: dissociation of depleted ion populations. A first MS/MS step is used to fragment isomers with relatively low appearance energy. The isomers with higher appearance energy are fragmented in a second MS/MS step of higher energy. The second approach is based on gas-phase H/D exchange experiments that result in a bimodal isotopic distribution for oligomers X(n)D(n+1) of which one distribution corresponds to a type of isomeric structure that exhibits H/D exchange behaviour and the other to an isomeric structure that does not exhibit H/D exchange behaviour. X is a difunctional anhydride of phthalic acid (P), 1,2-cyclohexanedicarboxylic acid (C), succinic acid (S) or glutaric acid (G). D in X(n)D(n+1) is a trifunctional diisopropanolamine and n the degree of polymerization. The type of isomeric structure that does not exhibit H/D exchange behaviour has a non-alternating monomer sequence that contains an amine bond with a relatively high proton affinity. The other isomeric structure that does exhibit H/D exchange behaviour has an alternating monomer sequence containing only amide and ester bonds with relatively low proton affinity. Oligomer structures were confirmed with additional MS(2) experiments after H/D exchange. H/D exchange experiments on the fragments obtained after MS(2) of the parent ion show that next to previously postulated mechanisms for the cleavage of the ester and amide bond another reaction pathway must be operational. A new mechanism is introduced to explain the H/D exchange behaviour of the fragments that requires a cleavage of the amide bonds only. Two types of fragments are formed by this mechanism. One type is protonated due to the cleavage of the amide bond whereas the other type has an oxazolonium ion structure due to the loss of an additional H(2)O.