Differentiation of 18O‐ and 16O‐rich layers in anodic alumina films by glow discharge time‐of‐flight mass spectroscopy

The performance of glow discharge time‐of‐flight mass spectrometry in isotopic differentiation is revealed using the distribution of oxygen isotopes ¹⁶O and ¹⁸O in barrier‐type anodic alumina films as a focus. Anodic alumina films comprising ¹⁸O‐rich layers of controlled thickness were formed by the appropriate combination of anodising of superpure aluminium in electrolytes enriched with ¹⁸O isotopes and of natural abundance of ¹⁸O isotopes. Analysis of the elemental depth profiles of selected ionic species, i.e. ¹⁶O¹⁸O, allowed determination of the locations of the ¹⁸O‐rich layers and the ¹⁸O/¹⁶O interface. Copyright © 2010 John Wiley & Sons, Ltd.     

Gas‐phase basicity of 2‐furaldehyde

2‐Furaldehyde (2‐FA), also known as furfural or 2‐furancarboxaldehyde, is an heterocyclic aldehyde that can be obtained from the thermal dehydration of pentose monosaccharides. This molecule can be considered as an important sustainable intermediate for the preparation of a great variety of chemicals, pharmaceuticals and furan‐based polymers. Despite the great importance of this molecule, its gas‐phase basicity (GB) has never been measured. In this work, the GB of 2‐FA was determined by the extended Cooks's kinetic method from electrospray ionization triple quadrupole tandem mass spectrometric experiments along with theoretical calculations. As expected, computational results identify the aldehydic oxygen atom of 2‐FA as the preferred protonation site. The geometries of O‐O‐cis and O‐O‐trans 2‐FA and of their six different protomers were calculated at the B3LYP/aug‐TZV(d,p) level of theory; proton affinity (PA) values were also calculated at the G3(MP2, CCSD(T)) level of theory. The experimental PA was estimated to be 847.9 ± 3.8 kJ mol^?1, the protonation entropy 115.1 ± 5.03 J mol^?1 K^?1 and the GB 813.6 ± 4.08 kJ mol^?1 at 298 K. From the PA value, a ΔH°_f of 533.0 ± 12.4 kJ mol^?1 for protonated 2‐FA was derived. Copyright © 2012 John Wiley & Sons, Ltd.     

Methods and limitations of ‘clumped’ CO2 isotope (Δ47) analysis by gas‐source isotope ratio mass spectrometry

The geochemistry of multiply substituted isotopologues (‘clumped‐isotope’ geochemistry) examines the abundances in natural materials of molecules, formula units or moieties that contain more than one rare isotope (e.g. ¹³C¹⁸O¹⁶O, ¹⁸O¹⁸O, ¹⁵N₂, ¹³C¹⁸O¹⁶O₂^2?). Such species form the basis of carbonate clumped‐isotope thermometry and undergo distinctive fractionations during a variety of natural processes, but initial reports have provided few details of their analysis. In this study, we present detailed data and arguments regarding the theoretical and practical limits of precision, methods of standardization, instrument linearity and related issues for clumped‐isotope analysis by dual‐inlet gas‐source isotope ratio mass spectrometry (IRMS). We demonstrate long‐term stability and subtenth per mil precision in 47/44 ratios for counting systems consisting of a Faraday cup registered through a 10¹² Ω resistor on three Thermo‐Finnigan 253 IRMS systems. Based on the analyses of heated CO₂ gases, which have a stochastic distribution of isotopes among possible isotopologues, we document and correct for (1) isotopic exchange among analyte CO₂ molecules and (2) subtle nonlinearity in the relationship between actual and measured 47/44 ratios. External precisions of ～0.01‰ are routinely achieved for measurements of the mass‐47 anomaly (a measure mostly of the abundance anomaly of ¹³C‐¹⁸O bonds) and follow counting statistics. The present technical limit to precision intrinsic to our methods and instrumentation is ～5 parts per million (ppm), whereas precisions of measurements of heterogeneous natural materials are more typically ～10 ppm (both 1 s.e.). These correspond to errors in carbonate clumped‐isotope thermometry of ±1.2 °C and ±2.4 °C, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

18O/16O ratio measurements of inorganic and organic materials by elemental analysis-pyrolysis-isotope ratio mass spectrometry continuous-flow techniques

We have used a high‐precision, easy, low‐cost and rapid method of oxygen isotope analysis applied to various O‐bearing matrices, organic and inorganic (sulfates, nitrates and phosphates), whose ¹⁸O/¹⁶O ratios had already been measured. It was first successfully applied to ¹⁸O analyses of natural and synthetic phosphate samples. The technique uses high‐temperature elemental analysis–pyrolysis (EA‐pyrolysis) interfaced in continuous‐flow mode to an isotope ratio mass spectrometry (IRMS) system. Using the same pyrolysis method we have been able to generate a single calibration curve for all those samples showing pyrolysis efficiencies independent of the type of matrix pyrolysed. We have also investigated this matrix‐dependent pyrolysis issue using a newly developed pyrolysis technique involving 'purge‐and‐trap' chromatography. As previously stated, silver phosphate being a very stable material, weakly hygroscopic and easily synthesized with predictable ¹⁸O/¹⁶O values, could be considered as a good candidate to become a reference material for the determination of ¹⁸O/¹⁶O ratios by EA‐pyrolysis‐IRMS. Copyright © 2011 John Wiley & Sons, Ltd.     

In‐time and in‐space tandem mass spectrometry to determine the metabolic profiling of flavonoids in a typical sweet cherry (Prunus avium L.) cultivar from Southern Italy

This paper presents a comprehensive analytical methodology, based on ‘in‐time’ and ‘in‐space’ tandem mass spectrometry (MS) techniques, to identify and quantify flavonoid compounds in a typical Italian sweet cherry cultivar (cv. Ferrovia). Five anthocyanins, four flavan‐3‐ols and nine flavonols were determined by means of hyphenated high‐performance liquid chromatography – multi‐stage MS (HPLC‐MSⁿ) analyses (MSⁿ up to MS⁴), among which quercetin‐3‐O‐rutinoside‐7‐O‐glucoside, kaempferol‐3‐O‐rutinoside‐7‐O‐glucoside, quercetin‐3‐O‐galactosyl‐rhamnoside and quercetin‐3‐O‐coumaroylglucoside were tentatively identified in sweet cherries for the first time. Ultrafast HPLC and tandem MS (UHPLC‐MS/MS) analyses through multiple reaction monitoring experiments showed that cyanidin‐3‐O‐rutinoside and cyanidin‐3‐O‐glucoside were the main anthocyanins of cv. Ferrovia at maturity. Moreover, consistent levels of catechin and epicatechin as well as quercetin‐3‐O‐rutinoside and kaempferol‐3‐O‐rutinoside were also found. Because flavonoids have been ascribed as potential health‐promoting compounds, gathered findings provide new insight into the knowledge of the quali‐quantitative profile of these phytochemicals into a widespread fruit such as sweet cherry. Copyright © 2014 John Wiley & Sons, Ltd.     

Elemental,Isotopic, and Pesticide Analysis of Wild and Cultivated Berries

ABSTRACT

The focus of this study was to investigate differences in isotopic, elemental, and trace pesticide concentrations of wild and cultivated berries from Transylvania. To emphasize differences based on geographical origin, stable isotopic ratios of ²H/¹H, ¹⁸O/¹⁶O, and ¹³C/¹²C were determined by isotopic ratio mass spectrometry. Elemental fingerprinting of berries was performed by inductively coupled plasma mass spectrometry. The determination of trace pesticides in berries was performed by gas chromatography–mass spectrometry. Differences between wild and cultivated berries were evaluated using multivariate statistical analysis. The results suggest that multielemental, isotopic, and trace pesticide fingerprinting is feasible for sample differentiation.     

Mass spectrometric method for the absolute calibration of the intramolecular nitrogen isotope distribution in nitrous oxide

A mass spectrometric method to determine the absolute intramolecular (position-dependent) nitrogen isotope ratios of nitrous oxide (N₂O) has been developed. It is based on the addition of different amounts of doubly labeled ¹⁵N₂O to an N₂O sample of the isotope ratio mass spectrometer reference gas, and subsequent measurement of the relative ion current ratios of species with mass 30, 31, 44, 45, and 46. All relevant quantities are measured by isotope ratio mass spectrometers, which means that the machines inherent high precision of the order of 10^–5 can be fully exploited. External determination of dilution factors with generally lower precision is avoided. The method itself can be implemented within a day, but a calibration of the oxygen and average nitrogen isotope ratios relative to a primary isotopic reference material of known absolute isotopic composition has to be performed separately. The underlying theoretical framework is explored in depth. The effect of interferences due to ¹⁴N¹⁵N¹⁶O and ¹⁵N¹⁴N¹⁶O in the ¹⁵N₂O sample and due to ¹⁵N ₂ ⁺ formation are fully accounted for in the calculation of the final position-dependent nitrogen isotope ratios. Considering all known statistical uncertainties of measured quantities and absolute isotope ratios of primary isotopic reference materials, we achieve an overall uncertainty of 0.9 (1). Using tropospheric N₂O as common reference point for intercomparison purposes, we find a substantially higher relative enrichment of ¹⁵N at the central nitrogen atom over ¹⁵N at the terminal nitrogen atom than measured previously for tropospheric N₂O based on a chemical conversion method: 46.3±1.4 as opposed to 18.7±2.2. However, our method depends critically on the absolute isotope ratios of the primary isotopic reference materials air–N₂ and VSMOW. If they are systematically wrong, our estimates will also necessarily be incorrect.     

Assessment of tandem mass spectrometry and high‐resolution mass spectrometry for the analysis of bupivacaine in plasma

Triple quadrupole mass spectrometers coupled with high performance liquid chromatography are workhorses in quantitative bioanalyses. They provide substantial benefits including reproducibility, sensitivity and selectivity for trace analysis. Selected reaction monitoring allows targeted assay development but datasets generated contain very limited information. Data mining and analysis of nontargeted high‐resolution mass spectrometry profiles of biological samples offer the opportunity to perform more exhaustive assessments, including quantitative and qualitative analysis. The objectives of this study were to test method precision and accuracy, to statistically compare bupivacaine drug concentration in real study samples and to verify if high‐resolution and accurate mass data collected in scan mode can actually permit retrospective data analysis, more specifically, extract metabolite related information. The precision and accuracy data presented using both instruments provided equivalent results. Overall, the accuracy ranged from 106.2 to 113.2% and the precision observed was from 1.0 to 3.7%. Statistical comparisons using a linear regression between both methods revealed a coefficient of determination (R²) of 0.9996 and a slope of 1.02, demonstrating a very strong correlation between the two methods. Individual sample comparison showed differences from ?4.5 to 1.6%, well within the accepted analytical error. Moreover, post‐acquisition extracted ion chromatograms at m/z 233.1648 ± 5 ppm (M ? 56) and m/z 305.2224 ± 5 ppm (M + 16) revealed the presence of desbutyl‐bupivacaine and three distinct hydroxylated bupivacaine metabolites. Post‐acquisition analysis allowed us to produce semi‐quantitative evaluations of the concentration–time profiles for bupicavaine metabolites. Copyright © 2015 John Wiley & Sons, Ltd.     

Application of gas chromatography–tandem mass spectrometry (GC/MS/MS) for the analysis of deuterium enrichment of water

Incorporation of deuterium from deuterium oxide (²H₂O) into biological components is a commonly used approach in metabolic studies. Determining the dilution of deuterium in the body water (BW) pool can be used to estimate body composition. We describe three sensitive GC/MS/MS methods to measure water enrichment in BW. Samples were reacted with NaOH and U‐¹³C₃‐acetone in an autosampler vial to promote deuterium exchange with U‐¹³C₃‐acetone hydrogens. Headspace injections were made of U‐¹³C₃‐acetone‐saturated air onto a 30‐m DB‐1MS column in electron impact‐mode. Subjects ingested 30 ml ²H₂O, and plasma samples were collected. BW was determined by standard equation. Dual‐energy X‐ray absorptiometry scans were performed to calculate body mass, body volume and bone mineral content. A four‐compartmental model was used to estimate body composition (fat and fat free mass). Full‐scan experiments generated an m/z 45 peak and to a lesser extent an m/z 61 peak. Product fragment ions further monitored included 45 and 46 using selected ion monitoring (Method1), the 61 > 45 and 62 > 46 transition using multiple reaction monitoring (MRM; Method2) and the neutral loss, 62 > 45, transition (Method3). MRM methods were optimized for collision energy (CE) and collision‐induced dissociation (CID) argon gas pressure with 6 eV CE and 1.5 mTorr CID gas being optimal. Method2 was used for final determination of ²H₂O enrichment of subjects because of lower natural background. We have developed a sensitive method to determine ²H₂O enrichment in BW to enable measurement of FM and FFM. Copyright © 2015 John Wiley & Sons, Ltd.     

Essential methodological improvements in the oxygen isotope ratio analysis of N‐containing organic compounds

The quantitative conversion of organically bound oxygen into CO, a prerequisite for the ¹⁸O/¹⁶O analysis of organic compounds, is generally performed by high‐temperature conversion in the presence of carbon at ～1450°C. Since this high‐temperature procedure demands complicated and expensive equipment, a lower temperature method that could be utilized on standard elemental analyzers was evaluated. By substituting glassy carbon with carbon black, the conversion temperature could be reduced to 1170°C. However, regardless of the temperature, N‐containing compounds yielded incorrect results, despite quantitative conversion of the bound oxygen into CO. We believe that the problems were partially caused by interfering gases produced by a secondary decomposition of N‐ and C‐containing polymers formed during the decomposition of the analyte. In order to overcome the interference, we replaced the gas chromatographic (GC) separation of CO and N₂ by reversible CO adsorption, yielding the possibility of collecting and purifying the CO more efficiently. After CO collection, the interfering gases were vented by means of a specific stream diverter, thus preventing them from entering the trap and the mass spectrometer. Simultaneously, a make‐up He flow was used to purge the gas‐specific trap before the desorption of the CO and its subsequent mass spectrometric analysis. Furthermore, the formation of interfering gases was reduced by the use of polyethylene as an additive for analytes with a N:O ratio greater than 1. These methodological modifications to the thermal conversion of N‐containing analytes, depending on their structure or O:N ratio, led to satisfactory results and showed that it was possible to optimize the conditions for their individual oxygen isotope ratio analysis, even at 1170°C. With these methodological modifications, correct and precise δ¹⁸O results were obtained on N‐containing analytes even at 1170°C. Differences from the expected standard values were below ±1‰ with standard deviations of the analysis <0.2‰. Copyright © 2010 John Wiley & Sons, Ltd.     

MALDI‐TOF Mass‐Spectrometry‐Based Versatile Method for the Characterization of Protein Kinases

We describe a MALDI‐TOF mass‐spectrometry‐based method that is rapid and versatile for the characterization of protein kinases and their inhibitors. We have designed new kinase substrates by the modification of common synthetic peptides, such as kemptide (LRRALS G), CaMKII substrate (KRQQS FDLF), erktide (ATGPLS PGPFGRR), abltide (EAIY AAPFAKKK), srctide (AEEEIY GEFEAKKKK), neurogranin (AAAKIQAS FRGHMARKK), and casein kinase I (CKI) substrate (RRKDLHDDEEDEAMS ITA). There are two fundamental points on which the proposed method is based to improve the mass‐spectrometric response: 1) mass tag technology by N‐derivatization through stable isotope labeling and 2) C‐terminal conjugation with tryptophanylarginine (WR). It was suggested that C‐terminal conjugation with the WR moiety enhances the ionization potency of these new substrates 1.5–13.7 times as much as those of the original peptides. We demonstrated, by using modified abltide (Ac‐EAIY AAPFAKKKWR‐NH₂), that WR conjugation at the C‐terminus in combination with stable‐isotope labeling at the N‐terminus allowed the quantitative assay of recombinant c‐Abl kinase in the presence of adenosine 5′‐triphosphate (ATP; K_M,ATP=18.6 μM and V_max=642 pmol min^?1 μg^?1). The present protocol made a simple and reliable inhibition assay of recombinant c‐Abl kinase by imatinib possible (IC_{50(recombinant)}=291 nM ; STI571, Gleevec; Novartis Pharma). Moreover, it was also demonstrated that this ATP noncompetitive inhibitor differentiates between two conformers of c‐Abl kinases: the phosphorylated active and dephosphorylated inactive forms (IC_{50(active form)}=1049 nM and IC_{50(inactive form)}=54 nM ). The merit of this approach is evident because the present protocol can be applied to the direct monitoring of the activities of living cell kinases by using cancer‐cell lines, such as mouse B16 melanoma cells and human lung cancer K562 cells. A multiple‐kinase assay that uses K562 cell lysate in the presence of seven new synthetic substrates made high‐throughput inhibitor profiling possible. It should be emphasized that this radioactive isotope‐free quantitative kinase assay will greatly accelerate the discovery of a new generation of potential kinase inhibitors that exhibit highly selective or unique inhibitory profiles.     

Increasing the accuracy of mass isotopomer analysis through calibration curves constructed using biologically synthesized compounds

Mass isotopomer analysis is an important technique to measure the production and flow of metabolites in living cells, tissues, and organisms. This technique depends on accurate quantifications of different mass isotopomers using mass spectrometry. Constructing calibration curves using standard samples is the most universal approach to convert raw mass spectrometry measurements into quantitative distributions of mass isotopomers. Calibration curve approach has been, however, of very limited use in comprehensive analyses of biological systems, mainly suffering from the lack of extensive range of standard samples with accurately known isotopic enrichment. Here, we present a biological method capable of synthesizing specifically labeled amino acids. These amino acids have well‐determined and estimable mass isotopomer distributions and thus can serve as standard samples. In this method, the bacterium strain Methylobacterium salsuginis sp. nov. was cultivated with partially ¹³C‐labeled methanol as the only carbon source to produce ¹³C‐enriched compounds. We show that the mass isotopomer distributions of the various biosynthesized amino acids are well determined and can be reasonably estimated based on proposed binomial approximation if the labeling state of the biomass reached an isotopic steady state. The interference of intramolecular inhomogeneity of ¹³C isotope abundances caused by biological isotope fractionation was eliminated by estimating average ¹³C isotope abundance. Further, the predictions are tested experimentally by mass spectrometry (MS) spectra of the labeled glycine, alanine, and aspartic acid. Most of the error in mass spectrometry measurements was less than 0.74 mol% in the test case, significantly reduced as compared with uncalibrated results, and this error is expected to be less than 0.4 mol% in real experiment as revealed by theoretical analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

A method combining SPITC and 18O labeling for simultaneous protein identification and relative quantification

The relative quantification and identification of proteins by matrix‐assisted laser desorption ionization time‐of‐flight MS is very important in /MS is very important in protein research and is usually conducted separately. Chemical N‐terminal derivatization with 4‐sulphophenyl isothiocyanate facilitates de novo sequencing analysis and accurate protein identification, while ¹⁸O labeling is simple, specific and widely applicable among the isotopic labeling methods used for relative quantification. In the present study, a method combining 4‐sulphophenyl isothiocyanate derivatization with ¹⁸O isotopic labeling was established to identify and quantify proteins simultaneously in one experiment. Reaction conditions were first optimized using a standard peptide (fibrin peptide) and tryptic peptides from the model protein (bovine serum albumin). Under the optimized conditions, these two independent labeling steps show good compatibility, and the linear relativity of quantification within the ten times dynamic range was stable as revealed by correlation coefficient analysis (R² value = 0.998); moreover, precursor peaks in MS/MS spectrum could provide accurate quantitative information, which is usually acquired from MS spectrum, enabling protein identification and quantification in a single MS/MS spectrum. Next, this method was applied to native peptides isolated from spider venoms. As expected, the de novo sequencing results of each peptide matched with the known sequence precisely, and the measured quantitative ratio of each peptide corresponded well with the theoretical ratio. Finally, complex protein mixtures of spider venoms from male and female species with unknown genome information were analyzed. Differentially expressed proteins were successfully identified, and their quantitative information was also accessed. Taken together, this protein identification and quantification method is simple, reliable and efficient, which has a good potential in the exploration of peptides/proteins from species with unknown genome. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization of phenolic compounds in green and red oak‐leaf lettuce cultivars by UHPLC‐DAD‐ESI‐QToF/MS using MSE scan mode

Lettuce (Lactuca sativa ) is one of the most popular leafy vegetables in the world and constitutes a major dietary source of phenolic compounds with health‐promoting properties. In particular, the demand for green and red oak‐leaf lettuces has considerably increased in the last years but few data on their polyphenol composition are available. Moreover, the usage of analytical edge technology can provide new structural information and allow the identification of unknown polyphenols. In the present study, the phenolic profiles of green and red oak‐leaf lettuce cultivars were exhaustively characterized by ultrahigh‐performance liquid chromatography (UHPLC) coupled online to diode array detection (DAD), electrospray ionization (ESI), and quadrupole time‐of‐flight mass spectrometry (QToF/MS), using the MS^E instrument acquisition mode for recording simultaneously exact masses of precursor and fragment ions. One hundred fifteen phenolic compounds were identified in the acidified hydromethanolic extract of freeze‐dried lettuce leaves. Forty‐eight of these compounds were tentatively identified for the first time in lettuce, and only 20 of them have been previously reported in oak‐leaf lettuce cultivars in literature. Both oak‐leaf lettuce cultivars presented similar phenolic composition, except for apigenin‐glucuronide and dihydroxybenzoic acid, only detected in the green cultivar; and for luteolin‐hydroxymalonylhexoside, an apigenin conjugate with molecular formula C₄₀H₅₄O₁₉ (monoisotopic MW = 838.3259 u ), cyanidin‐3‐O ‐glucoside, cyanidin‐3‐O ‐(3″‐O ‐malonyl)glucoside, cyanidin‐3‐O ‐(6″‐O ‐malonyl)glucoside, and cyanidin‐3‐O ‐(6″‐O ‐acetyl)glucoside, only found in the red cultivar. The UHPLC‐DAD‐ESI‐QToF/MS^E approach demonstrated to be a useful tool for the characterization of phenolic compounds in complex plant matrices.     

Interaction of [VIVO(acac)2] with Human Serum Transferrin and Albumin

[VO(acac)₂] is a remarkable vanadium compound and has potential as a therapeutic drug. It is important to clarify how it is transported in blood, but the reports addressing its binding to serum proteins have been contradictory. We use several spectroscopic and mass spectrometric techniques (ESI and MALDI‐TOF), small‐angle X‐ray scattering and size exclusion chromatography (SEC) to characterize solutions containing [VO(acac)₂] and either human serum apotransferrin (apoHTF) or albumin (HSA). DFT and modeling protein calculations are carried out to disclose the type of binding to apoHTF. The measured circular dichroism spectra, SEC and MALDI‐TOF data clearly prove that at least two VO–acac moieties may bind to apoHTF, most probably forming [V^IVO(acac)(apoHTF)] complexes with residues of the HTF binding sites. No indication of binding of [VO(acac)₂] to HSA is obtained. We conclude that V^IVO–acac species may be transported in blood by transferrin. At very low complex concentrations speciation calculations suggest that [(VO)(apoHTF)] species form.     

Infrared Spectroscopy of Dioxouranium(V) Complexes with Solvent Molecules: Effect of Reduction

UO₂⁺–solvent complexes having the general formula [UO₂(ROH)]⁺ (R=H, CH₃, C₂H₅, and n‐C₃H₇) are formed using electrospray ionization and stored in a Fourier transform ion cyclotron resonance mass spectrometer, where they are isolated by mass‐to‐charge ratio, and then photofragmented using a free‐electron laser scanning through the 10 μm region of the infrared spectrum. Asymmetric O=U=O stretching frequencies (ν₃) are measured over a very small range [from ～953 cm^?1 for H₂O to ～944 cm^?1 for n‐propanol (n‐PrOH)] for all four complexes, indicating that the nature of the alkyl group does not greatly affect the metal centre. The ν₃ values generally decrease with increasing nucleophilicity of the solvent, except for the methanol (MeOH)‐containing complex, which has a measured ν₃ value equal to that of the n‐PrOH‐containing complex. The ν₃ frequency values for these U(V) complexes are about 20 cm^?1 lower than those measured for isoelectronic U(VI) ion‐pair species containing analogous alkoxides. ν₃ values for the U(V) complexes are comparable to those for the anionic [UO₂(NO₃)₃]^? complex, and 40–70 cm^?1 lower than previously reported values for ligated uranyl(VI) dication complexes. The lower frequency is attributed to weakening of the O?U?O bonds by repulsion related to reduction of the U metal centre, which increases electron density in the antibonding π* orbitals of the uranyl moiety. Computational modelling of the ν₃ frequencies using the B3LYP and PBE functionals is in good agreement with the IRMPD measurements, in that the calculated values fall in a very small range and are within a few cm^?1 of measurements. The values generated using the LDA functional are slightly higher and substantially overestimate the trends. Subtleties in the trend in ν₃ frequencies for the H₂O–MeOH–EtOH–n‐PrOH series are not reproduced by the calculations, specifically for the MeOH complex, which has a lower than expected value.     

IRMPD Spectroscopy of a Protonated,Phosphorylated Dipeptide

The protonated, phosphorylated dipeptide [GpY+H]⁺ is characterized by mid‐infrared multiple‐photon dissociation (IRMPD) spectroscopy and quantum‐chemical calculations. The ions are generated in an external electrospray source and analyzed in a Fourier transform ion cyclotron resonance mass spectrometer, and their fragmentation is induced by resonant absorption of multiple photons emitted by a tunable free‐electron laser. The IRMPD spectra are recorded in the 900–1730 cm^?1 range and compared to the absorption spectra computed for the lowest energy structures. A detailed calibration of computational levels, including B3LYP‐D and coupled cluster, is carried out to obtain reliable relative energies of the low‐energy conformers. It turns out that a single structure can be invoked to assign the IRMPD spectrum. Protonation at the N terminus leads to the formation of a strong ionic hydrogen bond with the phosphate P?O group in all low‐energy structures. This leads to a P?O stretching frequency for [GpY+H]⁺ that is closer to that of [pS+H]⁺ than to that of [pY+H]⁺ and thus demonstrates the sensitivity of this mode to the phosphate environment. The COP phosphate ester stretching mode is confirmed to be an intrinsic diagnostic for identification of which type of amino acid is phosphorylated.     

18O distributions in porous anodic alumina by plasma profiling time‐of‐flight mass spectrometry and nuclear reaction analysis

A comparison is made between plasma profiling time‐of‐flight mass spectrometry (PP‐TOFMS) and nuclear reaction analysis (NRA) for depth profiling of ¹⁸O tracer in porous anodic oxide films on aluminum. The films were formed galvanostatically, for a range of times, using phosphoric acid electrolytes that were either enriched in ¹⁸O or of the natural isotopic concentration. The morphologies of the films were determined by electron microscopy. The findings from PP‐TOFMS and NRA reveal a partitioning of the tracer between the surface regions and buried layers of the films. However, a relatively high background of ¹⁶O in PP‐TOFMS prevents a reliable quantification of the concentration of ¹⁸O. Copyright © 2012 John Wiley & Sons, Ltd.     

Separation and Quantification of Water-Soluble Cellular Metabolites in Clostridium thermocellum using Liquid Chromatography-Isotope Dilution Tandem Mass Spectrometry

A new protocol for metabolomic studies was developed by combining liquid chromatography-tandem mass spectrometry and isotope dilution mass spectrometry with universal ¹³C labeled internal standards from Escherichia Coli. The multiple reaction monitoring mode of mass spectrometry was used for quantification. Forty-five water-soluble intracellular metabolites, including 20 amino acids, 16 organic acids (primarily from the tricarboxylic acid cycle), and 9 cofactors, were measured and 34 of them were successfully quantified using the ¹³C-labeled internal standards. The limit of detection, limit of quantification, precision, and linearity of the methods were evaluated. The methods were applied to the quantitative analysis of intracellular metabolites extracted from wild-type and ethanol-adapted strains of Clostridium thermocellum cultivated with and without ethanol stress, and all 34 metabolites including all 9 cofactors were successfully quantified. Further multivariate data analyses of the metabolic differences between wild-type and ethanol-adapted strains were performed on the quantitative data, which can help elucidate the metabolic mechanism behind ethanol adaptation in C. thermocellum.

Supplemental materials are available for this article. Go to the publisher's online edition of Analytical Letters to view the supplemental file.     

Differentiation of isomeric dinitrotoluenes and aminodinitrotoluenes using electrospray high resolution mass spectrometry

Explosive detection and identification play an important role in the environmental and forensic sciences. However, accurate identification of isomeric compounds remains a challenging task for current analytical methods. The combination of electrospray multistage mass spectrometry (ESI‐MSⁿ) and high resolution mass spectrometry (HRMS) is a powerful tool for the structure characterization of isomeric compounds. We show herein that resonant ion activation performed in a linear quadrupole ion trap allows the differentiation of dinitrotoluene isomers as well as aminodinitrotoluene isomers. The explosive‐related compounds: 2,4‐dinitrotoluene (2,4‐DNT), 2,6‐dinitrotoluene (2,6‐DNT), 2‐amino‐4,6‐dinitrotoluene (2A‐4,6‐DNT) and 4‐amino‐2,6‐dinitrotoluene (4A‐2,6‐DNT) were analyzed by ESI‐MS in the negative ion mode; they produced mainly deprotonated molecules [M ? H]^?. Subsequent low resolution MSⁿ experiments provided support for fragment ion assignments and determination of consecutive dissociation pathways. Resonant activation of deprotonated dinitrotoluene isomers gave different fragment ions according to the position of the nitro and amino groups on the toluene backbone. Fragment ion identification was bolstered by accurate mass measurements performed using Fourier transform ion cyclotron resonance mass spectrometry (FT‐ICR/MS). Notably, unexpected results were found from accurate mass measurements performed at high resolution for 2,6‐DNT where a 30‐Da loss was observed that corresponds to CH₂O departure instead of the expected isobaric NO^? loss. Moreover, 2,4‐DNT showed a diagnostic fragment ion at m/z 116, allowing the unambiguous distinction between 2,4‐ and 2,6‐DNT isomers. Here, CH₂O loss is hindered by the presence of an amino group in both 2A‐4,6‐DNT and 4A‐2,6‐DNT isomers, but nevertheless, these isomers showed significant differences in their fragmentation sequences, thus allowing their differentiation. DFT calculations were also performed to support experimental observations. Copyright © 2014 John Wiley & Sons, Ltd.