Exchangeable Hydrogen Research Articles

Accumulative Tritium Transfer from Water into Biosystems

The energy balance of hydrogen isotopes in H bonds of water and biomolecules results in accumulative tritium transfer from water into biomolecules. Tests of DNA dissolved in tritiated water and of maize or barley hydroponically grown in tritiated water confirm the increase. The primary hydration shell of DNA shows an accumulation factor of ~1.4, and the exchangeable hydrogens inside DNA show ~2. Logistic growth analyses of maize and barley reveal the intrinsic growth rates of tritium 1.3 and 1.2 times larger than that of hydrogen. The higher rate of tritium than hydrogen incorporation in solid biomatter is caused by the hydration shells, which constitute an intrinsic component of biomolecules.

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.

Probing the Structure of the Infectious Amyloid Form of the Prion-forming Domain of HET-s Using High Resolution Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry

The HET-s prion protein of Podospora anserina represents a valuable model system to study the structural basis of prion propagation. In this system, prion infectivity can be generated in vitro from a recombinant protein. We have previously identified the region of the HET-s protein involved in amyloid formation and prion propagation. Herein, we show that a recombinant peptide corresponding to the C-terminal prion-forming domain of HET-s (residues 218-289) displays infectivity. We used high resolution hydrogen/deuterium exchange analyzed by mass spectrometry to gain insight into the structural organization of this infectious amyloid form of the HET-s-(218-289) protein. Deuterium incorporation was analyzed by ion trap mass spectrometry for 76 peptides generated by pepsin proteolysis of HET-s-(218-289). By taking into account sequence overlaps in these peptides, a resolution ranging from 4-amino acids stretches to a single residue could be achieved. This approach allowed us to define highly protected regions alternating with more accessible segments along the HET-s-(218-289) sequence. The HET-s-(218-289) fibrils are thus likely to be organized as a succession of beta-sheet segments interrupted by short turns or short loops.

Fragmentation of Peptides in MALDI In-Source Decay Mediated by Hydrogen Radicals

In-source decay (ISD) in matrix-assisted laser desorption/ionization (MALDI) shares some similarities with the novel fragmentation technique electron capture dissociation (ECD). In both reactions, the otherwise strong N-C(alpha) bond is cleaved, forming fragment ions of the c and z types, while labile posttranslational modifications are preserved. Therefore, it is tempting to assume that ISD and ECD have some mechanistic aspects in common. Because electrons are present in the MALDI plume, we investigated the previously suggested possibility that ISD is a variation of ECD. However, experiments with peptides with only one site for efficient protonation revealed that ISD is not caused by electron capture. Instead, ICD seems to be induced by hydrogen atoms generated by a photochemical reaction of the matrix. We provide evidence for this reaction by hydrogen/deuterium exchange experiments with peptides containing a minimal number of exchangeable hydrogen atoms. The hydrogen atom model in ECD is indirectly supported by the proposed fragmentation mechanism for ISD, because our data suggest that hydrogen radicals can induce fragmentation by cleavage of the N-C(alpha) bond, independent from their origin.

Experimental Evidence of Differences in the Hydrogen-Bonded Structure of Concentrated Aqueous Solutions Involving Dl- and l-Alanine Molecules

Abstract Time-of-Flight (TOF) and steady state reactor neutron diffraction measurements have been carried out for aqueous 2.5 mol% Dl- and l-alanine solutions in order to investigate the differences in intermolecular hydrogen-bonded structure between solutions involving amino acid molecules with different optical activities. The observed difference functions, Δi(Q) and Δiinter(Q), between observed scattering cross sections for Dl- and l-alanine solutions with a D content of 96.1% exhibit the first peak located at Q = 2 Å−1 followed by oscillatory features extending up to the higher-Q region. The difference distribution function, Δg(r), obtained from the Fourier transform of Δi(Q), clearly indicates negative peaks at r = 2 and 2.5 Å and a positive one at r = 3.5 Å. Partial structure factors, aHH(Q), aXH(Q), and aXX(Q) (X: O, N, C, HM, and HM′, where HM and HM′ denote methyl and methine hydrogen atoms within the alanine molecule, respectively) for 2.5 mol% Dl-alanine solutions are successfully determined from combined analyses of intermolecular interference terms observed for solutions with 96.1, 66.0, and 35.9% exchangeable deuterium content. The nearest neighbor O···Hex and Hex···Hex (Hex: exchangeable hydrogen atom) distances are determined from the least squares fit of the observed partial structure factors, 1.90(1) and 2.48(1) Å, respectively. These values correspond to the positions of negative peaks observed in the present Δg(r) function. The least squares fitting analysis of the observed Δi(Q) revealed that the difference in the coordination number of the nearest neighbor O···Hex and Hex···Hex interactions between the Dl- and l-alanine solutions are −0.031(5) and −0.072(5), respectively. It is concluded that the intermolecular hydrogen bonds among solvent water molecules in the Dl-alanine solution are ca. 2% weaker than those in the l-alanine solution.

Reaction Dynamics of H2O+ (D2O+) + NH3 Studied with Crossed Molecular Beams and Density Functional Theory Calculations

The charge transfer and proton transfer reactions between H 2 O + (D 2 O + ) and NH 3 were studied at collision energies below I eV using the crossed molecular beam technique and density functional theory (DFT) calculations. The reaction products include NH 3 D + formed by deuterium ion transfer, NH 3 + produced by charge transfer, and NH 2 D + formed by charge transfer with H/D exchange. These three products are formed in the ratio of 1.0:2.0:1.2, respectively. The center of mass flux distributions of the product ions for all three reaction channels exhibit asymmetry, with maxima close to the velocity and direction of the precursor ammonia beam, characteristic of direct reactions. The internal energy distributions of the products of charge transfer are independent of collision energy, are very narrow (∼0.55 eV) and are peaked at the reaction exothermicity of 2.55 eV. The NH 2 D + products have very similar distributions, peaking at the reaction exothermicity and having widths of ∼0.70 eV. The mechanisms for these reaction channels are discussed on the basis of the DFT calculated potential energy surfaces, which identify three electrostatically bound complexes of the form [H 2 O.NH 3 ] + that mediate reaction. Theory suggests that H/D exchange to form NH 2 D + may occur through the complex NH 3 D + ...OD, in which an internal rotation of the NH 3 D + unit exchanges hydrogen and deuterium atoms. The large fraction of NH 2 D + reaction products observed indicates that this exchange takes place at a rate nearly 3 orders of magnitude faster than the predictions of statistical theory.

In vitro metabolism of ethoxidine by human CYP1A1 and rat microsomes: identification of metabolites by high-performance liquid chromatography combined with electrospray tandem mass spectrometry and accurate mass measurements by time-of-flight mass spectrometry.

Ethoxidine (N-methyl-12-ethoxy-2,3,8,9-tetramethoxybenzo[c]phenanthridinium methylsulfonate salt) is a synthetic 2-methoxy-12-ethoxy derivative of the natural alkaloid fagaronine. This new inhibitor of DNA-topoisomerase I is considered as a potential antitumor agent with higher in vitro activity than fagaronine. In order to further improve the efficiency of ethoxidine, its in vitro biotransformation by hepatic monooxygenases and the structures of its metabolites were investigated by high-performance liquid chromatography (HPLC) combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) and accurate mass measurement by time-of-flight mass spectrometry (TOFMS). When ethoxidine was incubated with BNF-treated rat liver microsomes or with cells expressing different recombinant human cytochrome P450, the same four ethoxidine metabolites (m(1)-m(4)) were detected and were formed exclusively by CYP1A1. The structures of these metabolites were assigned from ESI-MS/MS mass spectra and compared with those of ethoxidine derivatives. Accurate mass measurements of in-source ESI-TOFMS fragment ions exhibited successive neutral losses of C(2)H(4) and CO for ethoxidine and its metabolites. Whereas a 15 Da loss (methyl radical) was observed for the metabolites m(1)-m(4) containing a quaternary ammonium group, a 16 Da loss (methane) was observed for ethoxidine and could have resulted from the presence of two methoxy groups at adjacent positions (C-2 and C-3). The proposed oxidative modifications of ethoxidine were further confirmed by determination of the number of exchangeable hydrogen atoms and by the proposed elemental compositions of the metabolites based on accurate mass measurements by TOFMS. Two major metabolites resulted from O-demethylation of ethoxidine; one was tentatively identified as 12-ethoxyfagaronine (m(3)) and the second as an O-demethylated ethoxidine isomer (m(4)). Two polar metabolites were shown to be O-demethylated (m(1)) and hydroxylated (m(2)) derivatives of 12-ethoxyfagaronine. When 12-ethoxyfagaronine was incubated under the same conditions as ethoxidine, m(2) was formed, thus supporting the proposal that 12-ethoxyfagaronine is the primary oxidative product of ethoxidine.

Process Synthesis: Design for Improved Effectiveness

This paper sets out the motivation for using process synthesis techniques that have been developed over the past 25 years. These techniques enable developing countries to design processes that bypass the incremental improvement route. The most developed aspects of process synthesis will be discussed with particular focus on pinch technology. The technology gives emphases on both energy and capital cost targets for heat exchanger networks (HENs) and on the design techniques developed to meet these targets. A retrofit path can be identified and costed to improve on an existing process, and yield a design target to meet specified investment limits. These techniques have been applied for use in mass exchange networks (MENs), water optimization, hydrogen and oxygen usage, and production scheduling. One of the most significant developments in these areas has been the impact diagram, which shows reduction in environmental impact versus investment. Use of these techniques willlead to processes that are more effective in their use of resources and are, therefore, more sustainable.

Evaluation of metal concentrations in edible vegetables grown in compost amended soil

Agricultural uses of compost usually have a positive effect on the yield of vegetable crops for human consumption. However, compost that contains heavy metals can transfer these components to soils and plants. To evaluate the contamination levels of metals in soil, compost, and edible vegetables, the Mn, Zn, Pb, Cd, Cu, and Ni total contents were measured. Metal availability in soils, as well as other variables – the pH, CEC (cation exchange capacity), total nitrogen, organic carbon, particle size distribution and mineralogy of the clay fraction – were examined in the soil samples. The analysed compost samples were produced from urban solid waste, cattle manure, and edible vegetable and tree pruning residues. The values of pH, CEC, total nitrogen, organic matter, exchangeable hydrogen and carboxylic groups were measured in the compost samples. Of the six metals examined in the soils, in general, Mn and Zn attained the highest concentrations, followed by Cu. Relatively high Mn, Zn, Cu, Cd and Pb concentrations were found in the soils. Metal concentrations extracted with DTPA were below the critical levels in soils, i.e. the levels above which toxicity is likely. In general, Zn, Pb, Cd, Cu and Ni concentrations in compost were lower than those reported by other workers, while Mn levels were within the range for this metal in compost. The results showed that there was an effect of the vegetable type (p < 0.01) for all the parameters examined. High Pb concentrations were found in lettuce and chive as compared with the tolerance limit for this metal in fresh vegetables in Brazil. Cadmium concentrations were also enhanced in the fresh vegetables compared with the typical concentrations of metals in plants. Zinc, Cu, Cd, and Ni concentrations were lower than the tolerance limits established for foods by the Brazilian legislation.

Direct detection of a hydrogen ligand in the [NiFe] center of the regulatory H2-sensing hydrogenase from Ralstonia eutropha in its reduced state by HYSCORE and ENDOR spectroscopy.

The regulatory H2-sensing [NiFe] hydrogenase of the beta-proteobacterium Ralstonia eutropha displays an Ni-C "active" state after reduction with H2 that is very similar to the reduced Ni-C state of standard [NiFe] hydrogenases. Pulse electron nuclear double resonance (ENDOR) and four-pulse ESEEM (hyperfine sublevel correlation, HYSCORE) spectroscopy are applied to obtain structural information on this state via detection of the electron-nuclear hyperfine coupling constants. Two proton hyperfine couplings are determined by analysis of ENDOR spectra recorded over the full magnetic field range of the EPR spectrum. These are associated with nonexchangeable protons and belong to the beta-CH(2) protons of a bridging cysteine of the NiFe center. The signals of a third proton exhibit a large anisotropic coupling (Ax = 18.4 MHz, Ay = -10.8 MHz, Az = -18 MHz). They disappear from the 1H region of the ENDOR spectra after exchange of H2O with 2H2O and activation with 2H2 instead of H2 gas. They reappear in the 2H region of the ENDOR and HYSCORE spectra. Based on a comparison with the spectroscopically similar [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F, for which the g-tensor orientation of the Ni-C state with respect to the crystal structure is known (Foerster et al. J. Am. Chem. Soc. 2003, 125, 83-93), an assignment of the 1H hyperfine couplings is proposed. The exchangeable proton resides in a bridging position between the Ni and Fe and is assigned to a formal hydride ion. After illumination at low temperature (T = 10 K), the Ni-L state is formed. For the Ni-L state, the strong hyperfine coupling observed for the exchangeable hydrogen in Ni-C is lost, indicating a cleavage of the metal-hydride bond(s). These experiments give first direct information on the position of hydrogen binding in the active NiFe center of the regulatory hydrogenase. It is proposed that such a binding situation is also present in the active Ni-C state of standard hydrogenases.

Comparative equilibration and online technique for determination of non-exchangeable hydrogen of keratins for use in animal migration studies

Stable hydrogen-isotope ratios (δD) of keratin provide a novel means for tracking geographical movements of birds and other species. Here we describe a rapid, low cost, analytical approach to facilitate online continuous-flow isotope-ratio mass spectrometry (CF-IRMS) δD analyses of keratins (120-160 samples per day) through the use of calibrated keratin working standards and "comparative equilibration" to correct for the effects of moisture on exchangeable hydrogen. It is anticipated that this analytical approach and CF-IRMS will greatly aid in providing cost effective and directly comparable δD results on keratins and feathers among various laboratories and researchers involved in animal migration studies.

Neutron diffraction studies on aqueous solutions of glucose

Neutron diffraction experiments were carried out at ambient temperature on aqueous solutions of glucose at concentrations of 1, 3, and 5 molal. The difference methods of neutron diffraction and isotopic substitution were used to determine various structure factors associated with all the exchangeable hydrogen atoms of the solutions. The results imply that the hydration of the glucose molecule is not concentration sensitive. It is also found that the average water structure itself is relatively unperturbed by the presence of glucose.

A survey of Sigatoka leaf disease (Mycosphaerella musicola Leach) of banana and soil calcium levels in North Queensland

Annual averages of lesion development of yellow Sigatoka caused by Mycosphaerella musicola were calculated from surveys conducted at 14-day intervals for 57 banana sites in the North Queensland production region situated in the wet tropics between Cardwell and Innisfail. Soil up to 250 mm depth was sampled from sites between March 2000 and September 2001. Soil parameters were compared with 12 months of leaf disease data averaged centrally about the time the soil sample was taken. The 4 different formulas for calcium expression tested all proved to be significant predictors of disease levels. The strongest relationship (r2 = 0.229) between soil calcium levels and disease levels was obtained from a formula based on an Albrecht interpretation of the soil test data, which includes a pH-derived estimation of exchangeable hydrogen in the denominator. A Chi square analysis on a model incorporating calcium and soil boron was statistically significant (P&lt;0.001). The model combining calcium and boron levels explained more variation in disease levels than calcium alone. Variation in other factors influencing disease such as fungicide program, weather conditions, soil moisture conditions and crop stage may account for variation in disease levels at low calcium sites.

H/D exchange of gas phase bradykinin ions in a linear quadrupole ion trap

The gas phase H/D exchange reaction of bradykinin ions, as well as fragment ions of bradykinin generated through collisions in an orifice skimmer region, have been studied with a linear quadrupole ion trap (LIT) reflectron time-of-flight (rTOF) mass spectrometer system. The reaction in the trap takes only tens of seconds at a pressure of few mTorr of D 2O or CD 3OD. The exchange rate and hydrogen exchange level are not sensitive to the trapping q value over a broad range, provided q is not close to the stability boundary (q = 0.908). The relative rates and hydrogen exchange levels of protonated and sodiated +1 and +2 ions are similar to those observed previously by others with a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer system. The doubly and triply protonated ions show multimodal isotopic distributions, suggesting the presence of several different conformations. The y fragment ions show greater exchange rates and levels than a or b ions, and when water or ammonia is lost from the fragment ions, no exchange is observed.

Simulations of the 1 H electron spin echo–electron nuclear double resonance and 2 H electron spin echo envelope modulation spectra of exchangeable hydrogen nuclei coupled to the S 2 –state photosystem II manganese cluster

The pulsed EPR methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the proximity of exchangeable hydrogens around the paramagnetic S(2)-state Mn cluster of the photosystem II oxygen-evolving complex. Although ESEEM and ESE-ENDOR are both pulsed electron paramagnetic resonance techniques, the specific mechanisms by which nuclear spin transitions are observed are quite different. We are able to generate good simulations of both (1)H ESE-ENDOR and (2)H ESEEM signatures of exchangeable hydrogens at the S(2)-state cluster. The convergence of simulation parameters for both methods provides a high degree of confidence in the simulations. Several exchangeable protons-deuterons with strong dipolar couplings are observed. In the simulations, two of the close ( approximately 2.5 A) hydrogen nuclei exhibit strong isotropic couplings and are therefore most probably associated with direct substrate ligation to paramagnetic Mn. Another two of the close ( approximately 2.7 A) hydrogen nuclei show no isotropic couplings and are therefore most probably not contained in Mn ligands. We suggest that these proximal hydrogens may be associated with a Ca(2+)-bound substrate, as indicated in recent mechanistic proposals for O(2) formation.

Complementary use of ion trap/time-of-flight mass spectrometry in combination with capillary high-pressure liquid chromatography: early characterization of in vivo metabolites of the cathepsin K inhibitor NVP-AAV490 in rat

Cathepsin K is a cysteine proteinase, primarily expressed in osteoclasts, which has a strong collagenolytic activity and plays an essential role involved in bone matrix degradation. Its inhibition could provide a novel approach to the treatment and prevention of osteoporosis. One structural class of lead compounds in our cathepsin K inhibitors program is based on an arylaminoethyl amide scaffold, which has potential metabolic weak points that might be stabilized by appropriate chemical modification(s). For the identification of potential metabolic “soft spots” and the rational design of improved derivatives, early biotransformation of a potent arylaminoethyl amide cathepsin K inhibitor (NVP-AAV490-NX) was investigated in plasma, urine and liver homogenates of rats after intravenous bolus administration of 10 mg/kg. The detection and identification of metabolites was achieved by high-resolution mass spectrometry (time-of-flight MS) and multi-dimensional mass spectrometry (ion trap MS). Both mass spectrometers were combined with reversed-phase capillary high-performance liquid chromatography columns. It was demonstrated that both mass analyzers complement each other and that, even in the sub-nanogram range, the resulting set of MS data can be successfully used to elucidate most of the metabolic changes unambiguously, solely by mass spectrometric techniques. The proposed metabolite structures were additionally corroborated by exact mass measurement of the protonated molecular ions to confirm the predicted elemental composition, by determination of the number of the exchangeable hydrogen atoms replacing water against deuterium oxide as mobile phase and, in one case, by an MS 3 product ion experiment in order to elucidate the site of conjugation.

Matrix Infrared Spectra and DFT Calculations of the Reactive MHx (x = 1, 2, and 3), (H2)MH2, MH2+, and MH4- (M = Sc, Y, and La) Species

Laser-ablated Sc, Y, and La react with molecular hydrogen to give MH, MH2+, MH2, MH3, and MH4- (M = Sc, Y, and La) during condensation in excess argon for characterization by matrix infrared spectroscopy. Annealing forms the dihydrogen complex (H2)MH2, which can be reduced to MH4- by electron capture. The (HD)MHD complex exchanges hydrogen positions on broadband photolysis to form primarily the (D2)MH2 complex. Doping the samples with CCl4 to capture ablated electrons markedly increases the MH2+ infrared band intensities and decreases the MH4- absorptions. Further annealing produces higher (H2)2MH2 complexes, which also exchange hydrogen positions. The reaction products are identified by deuterium and deuterium hydride isotopic substitution. DFT and MP2 theoretical calculations are employed to predict geometries and vibrational frequencies of these novel molecules, complexes, anions, and cations. Charged species from laser-ablation contribute more to the spectra of group 3 reaction products than for any o...

Hydrogen/deuterium exchange and aggregation of a polyvaline and a polyleucine alpha-helix investigated by matrix-assisted laser desorption ionization mass spectrometry.

The membrane-associated pulmonary surfactant protein C (SP-C), containing a polyvaline alpha-helix, and a synthetic SP-C analogue with a polyleucine helix (SP-C(Leu)) were studied by hydrogen/deuterium exchange matrix-assisted laser desorption ionization (MALDI) mass spectrometry. SP-C, but not SP-C(Leu), formed abundant amyloid fibrils under experimental conditions. In CD(3)OD/D(2)O, 91:9 (v/v), containing 2 mM ammonium acetate, SP-C(Leu) and SP-C exchanged 40% of their exchangeable hydrogens within 1 min. This corresponds to exchange of labile side-chain hydrogen atoms, hydrogens on the N- and C-terminal heteroatoms, and amide hydrogen atoms in the unstructured N-terminal regions. After approximately 300 h, four exchangeable hydrogen atoms in SP-C(Leu) and 10 in SP-C remained unexchanged. During this time period the ion current corresponding to singly charged SP-C decreased to <10% of the initial value due to the formation of insoluble aggregates that are not detected by MALDI mass spectrometry. In contrast, the ion current for SP-C(Leu) was maintained over this time period, although the peptides were incubated together. In combination, hydrogen/deuterium exchange and aggregation data indicate that the polyleucine peptide refolds into a helix after opening, while the unfolded polyvaline peptide forms insoluble beta-sheet aggregates rather than refolding into a helix. The SP-C helix, but not the SP-C(Leu) helix, is thus in a metastable state, which may contribute to the recently observed tendency of SP-C and its precursor to misfold and aggregate in vivo.

Isotopically exchangeable organic hydrogen in coal relates to thermal maturity and maceral composition

Hydrogen isotopic exchangeability (H ex) and δD n values of non-exchangeable organic hydrogen were investigated in coal kerogens ranging in rank from lignite to graphite. The relative abundance of H ex is highest in lignite with about 18% of total hydrogen being exchangeable, and decreases to around 2.5% in coals with R o of 1.7 to ca. 5.7%. At still higher rank ( R o > 6%), H ex increases slightly, although the abundance of total hydrogen decreases. δD n is influenced by original biochemical D/H ratios and by thermal maturation in contact with water. Therefore, δD n does not show an overall consistent trend with maturity.

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 (D 2O) 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 H 2O or D 2O 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 product-ion-spectra helped to determine the location of the exchanged protons and assisted in the assignment of the site(s) of modification for each metabolite.