Visualization of the hydrogen desorption process from ferrite,pearlite, and graphite by secondary ion mass spectrometry

The distribution and desorption processes of hydrogen and deuterium have been visualized by secondary ion mass spectrometry (SIMS). The present article deals with four principal points: (1) visualizing the hydrogen distribution, (2) visualizing the hydrogen desorption process from each metallurgical microstructure under various holding times at 25 °C, (3) visualizing the hydrogen desorption process during heating, and (4) determining the correspondence between desorption profiles and desorption sites. A spheroidal graphite cast iron specimen was prepared for visualizing hydrogen, since it consists of basic microstructures of steels such as ferrite and pearlite. Hydrogen and deuterium were occluded into the cast iron. The amount of hydrogen and the existing states of hydrogen in the cast iron were analyzed by thermal desorption spectrometry (TDS). The TDS analyses show that the hydrogen desorption has two peaks, namely, the low- and high-temperature peaks corresponding to trap activation energies of 21.6 and 105.8 kJ/mol, respectively. The SIMS analyses of the specimen cooled after heating to 100 °C, 200 °C, and 300 °C reveal that the hydrogen desorbs from the ferrite after heating to 100 °C, from the pearlite and the interfaces between the ferrite and the graphite after heating to 200 °C, and from the pearlite after heating to 300 °C. The graphite remains, trapping hydrogen after heating to 300 °C. On the basis of TDS and SIMS results, the relationship between the desorption profile and desorption sites was identified; that is, the low-temperature peak corresponds to ferrite, pearlite, and graphite/ferrite interfaces, while the high-temperature peak corresponds to graphite.     

Analysis of organic reactions by thin layer chromatography combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The products of a wide variety of organic reactions were rapidly identified by their masses through a combination of thin layer chromatography (TLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Crude mixtures of peptides and glycopeptides, complex carbohydrate reactions, and a classical organic reaction were analysed using the following standard protocol. The components of the reaction mixtures were first separated on the TLC plate, scraped off, extracted and analysed by MALDI-TOFMS. The technique used is easy and applicable to most organic reactions, becoming a very powerful technique in reaction optimization once composition and identity of TLC spots have been established by MS. Moreover, the TLC/MALDI-TOFMS method was used to identify low molecular weight compounds with masses within the matrix region (100-500 u), a goal which is normally difficult to achieve. We successfully detected low molecular weight compounds by suppression of the matrix peaks using a relatively low matrix:analyte ratio (15:1 or lower). Doping both matrix and analyte solutions with [Cs]+ ions resulted in suppression of both [Na]+ and [K]+ peaks, thus enhancing the spectral signals and making identification of low molecular weight compounds more facile.     

ESI/ion trap/ion mobility/time-of-flight mass spectrometry for rapid and sensitive analysis of biomolecular mixtures

An ion trap/ion mobility/time-of-flight mass spectrometry technique is shown to be a rapid and sensitive means of analyzing peptide/protein mixtures. In this approach, an ion trap is used to accumulate ions that have been electrosprayed from a mixture into concentrated packets. The ion packets are injected into a drift tube where components of the mixture are separated based on differences in mobility through a buffer gas. Ions that exit the drift tube are dispersed in a time-of-flight mass spectrometer for mass-to-charge (m/z) determination. The gas-phase separation strategy reduces congestion in the mass spectrum, and experimental mobilities complement m/z measurements in assigning peaks. Examples of the application of the approach to identification of peptides (from tryptic digests) and to separation of charge-state distributions from electrospray of a mixture containing ubiquitin and myoglobin are presented. Most peptides that are observed from tryptic digests of proteins such as cytochrome c and myoglobin can be identified from data that are acquired in under 1 min; studies of mixtures with known compositions indicate that detection limits are approximately 0.5-3 pmol for individual components. Factors that may influence the distributions that are observed, such as storage time in the trap, injection voltages used for the mobility experiment, and variations in ion cross section with charge state, are discussed.     

Studies on the determination of surface deuterium in AISI 1062, 4037, and 4140 steels by secondary ion mass spectrometry

The concentration of deuterium at the surface of cathodically charged high strength steels AISI 1062, 4037, and 4140 has been determined by secondary ion mass spectrometry (SIMS). The beneficial effects of pickling in NAP (a mixture of nitric, acetic, and phosphoric acids) to remove surfacebound deuterium have been observed. formerly with AMCA International Limited, Kanata, ON, Canada     

Endgroup analysis of polyethylene glycol polymers by matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry

Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) by external injection of matrix-assisted laser desorbed and ionized (MALDI) polymers offers good possibilities for characterization of low molecular weight homopolymers (MW range up to 10 kDa). The molecular masses of the molecular weight distribution (MWD) components of underivatized and derivatized (dimethyl, dipropyl, dibutyl and diacetyl) polyethylene glycol (PEG) 1000 and 4000 were measured by MALDI-FTICR-MS. These measurements have been performed using a commercial FTICR spectrometer with a home-built external ion source. MALDI of the samples with a 2,5-dihydroxybenzoic acid matrix in a 1000:1 matrix-to-analyte molar ratio produces sodiated molecules in a sufficient yield to trap the ions in the ICR cell. The masses of the molecular weight distribution of PEG components were measured in broad-band mode with a mass accuracy of < 5 ppm in the mass range around 1000 u and within 40 ppm accuracy around 4000 u. From these measurements, the endgroup mass of the polymer was determined by correlation of the measured component mass with the degree of polymerization. The masses of the PEG endgroups have been determined within a deviation of 3-10 millimass units for the PEG1000 derivatives and 10-100 millimass units for the PEG4000 derivatives, thus confirming the identity of the distal parts of the model compounds.     

Pre-steady state kinetic analysis of an enzymatic reaction monitored by time-resolved electrospray ionization mass spectrometry

For the first time, the new technique of time-resolved electrospray ionization mass spectrometry (ESI-MS) has been used to accurately measure the pre-steady state kinetics of an enzymatic reaction by monitoring a transient enzyme intermediate. The enzyme used to illustrate this approach, Bacillus circulans xylanase, is a retaining glycosidase that hydrolyzes xylan or beta-xylobiosides through a double-displacement mechanism involving a covalent xylobiosyl-enzyme intermediate. A low steady state level of this intermediate formed during the hydrolysis of 2,5-dinitrophenyl beta-d-xylobioside was detected by time-resolved ESI-MS. The low concentration of this intermediate and its rate of formation did not permit pre-steady state kinetic analysis. By contrast, the covalent intermediate accumulates fully when the Tyr80Phe mutant hydrolyzes the same substrate. Using time-resolved ESI-MS, the pre-steady state kinetic parameters for the formation of the covalent intermediate in the mutant xylanase have been determined. The kinetic data are in agreement with those determined by monitoring the release of 2, 5-dinitrophenol with stopped-flow UV-vis spectroscopy. This demonstrates that time-resolved ESI-MS can be used to accurately monitor the pre-steady state kinetics of enzymatic reactions, with the advantage of identifying transient enzyme intermediates by their mass.     

The quantitative analysis of inhalational anaesthetics in forensic samples by gas chromatography/mass spectrometry/selected ion monitoring

In 1990 Kapolowé was, without a doubt, the site of the only surgical centre in Zaire dealing with handicaps which developed in as an after-effect of leprosy. It would be useful to explain the hazards involved in such a venture for reasons which do not pertain to medicine but, rather, to particularly trying socio-political circumstances. The best surgical expertise was thrown out for political reasons. Insecurity and economic hardships practically halted movement and, consequently, the wider application of such expertise. During a mission in 1994, there was a partial resumption of activities. The surgical team was reinstalled and made operational. It had been possible to state that multidrug therapy (MDT) had always ensured that the disabled leprosy patients, living in groups, and treated before 1990 under regular supervision, did not experience serious relapses. That fact corroborates earlier information relating particularly to surgical decompression. Although most of them were able to resume a certain measure of professional activity, social factors must still be borne in mind and the concept of partial permanent disability must be applied.     

Improved ternary matrices for the analysis of ferri-pyoverdins by fast atom bombardment mass spectrometry

The use of explosives as matrices, a principle used previously in plasma desorption mass spectrometry, was applied to fast atom bombardment mass spectrometry (FABMS). Ternary matrices were prepared by the addition of picric acid and related compounds to the binary matrix routinely used for the analysis of peptides, viz. thioglycerol-dithiodiethanol. With the new matrices, the spectra of three ferri-pyoverdins (iron-siderophore complexes which are often difficult to analyse by FABMS with standard matrices) showed a significant increase in both intensity and duration of the ion current. They present a valuable tool for the analysis of problematic analytes and for collision-induced dissociation MS/MS experiments.     

Differentiation of lysine/glutamine in peptide sequence analysis by electrospray ionization sequential mass spectrometry coupled with a quadrupole ion trap

Electrospray ionization coupled to a quadrupole ion trap mass spectrometer is used to differentiate between the isobaric amino acids lysine and glutamine in sequence analysis of peptides. Collision-induced dissociation is used for fragmentation. Several isobaric peptides with one or more lysines or glutamines at different positions were investigated. The ambiguous amino acid either in the peptide chain or at the C- or N-terminus can be clearly identified based on specific side chain fragment ions resulting from MS3 or MS4 of B- and Y"-fragment ions.     

Quantitative measurement of BN50727 in human plasma and urine by combined liquid chromatography/negative ion chemical ionization mass spectrometry using a particle beam interface

A new sensitive assay has been developed for the quantitative measurement of BN50727 at the picomole level in human plasma and urine. The drug and the internal standard (BN50788) were measured by combined liquid chromatography/negative ion chemical ionization mass spectrometry with methane as the reagent gas. A simple solid-liquid extraction procedure was used to isolate BN50727 from the complex biological matrices. The mass spectrometer was tuned to monitor the intense and stable ion at m/z 333 which was generated in the ion source by a dissociative capture process. This assay was performed with 1 ml of plasma or 0.1 ml of urine and the quantification limit of the method was statistically calculated as 1 ng ml-1. The very low relative standard deviations and mean percentages of error calculated during the different within-day or between-day repeatability assays have clearly demonstrated the ruggedness of the technique for the routine determination of BN50727 in biological fluids. Some preliminary results on the pharmacokinetics of the drug are presented to illustrate the applicability of this powerful liquid chromatographic/mass spectrometric method.     

Microscale analysis of glycosphingolipids by TLC blotting/secondary ion mass spectrometry: a novel blood group A-active glycosphingolipid and changes in glycosphingolipid expression in rat mammary tumour cells with different metastatic potentials

The glycosphingolipid compositions of rat mammary tumour cell lines with different metastatic potentials for the lung [a parental tumour cell line (MTC) and its subclones MTLn2 (a non metastatic subclone) and MTLn3 (a subclone with high metastatic potential to the lung)] were studied using a newly developed TLC blotting/secondary ion mass spectrometry system and crude glycosphingolipids obtained from 0.5-1 x 10(7) cells of each cell line. GM3 and GM2 were the major components of the MTC cell line, but they were very minor components in the MTLn2 and MTLn3 cell lines, GDla being the major ganglioside HexNAc-fucosyl-GMla was found in the MTLn2 cells by the TLC blotting/SIMS method, and the terminal sugar linkage was shown to be a blood group A-type structure by immunostaining. These findings suggest that the ganglioside is a novel type of blood group A-active ganglioside, GalNAc alpha 1-3(fuc alpha 1 -2)GMla. No blood group A-active lipid was present in MTLn3 cells, whereas Hex-GMla and neutral glycosphingolipids with more than 5 sugar residues were.     

Structural analysis and quantitative evaluation of the modifications produced in human hemoglobin by methyl bromide using mass spectrometry and Edman degradation

The present study reports a procedure developed for the identification and quantitative analysis of the adducts formed by interaction of methyl bromide with human hemoglobin, based on combined analysis by electrospray mass spectrometry and automated Edman degradation of either intact globin chains or tryptic peptides of globin chains. The procedure has allowed identification of the reactive sites in human hemoglobin, and has been applied to the analysis of samples modified in vitro by methyl bromide. The results obtained represent the basis for the complete structural characterization of the modified hemoglobin and demonstrate the usefulness of the proposed analytical approach for the evaluation of the degree of alkylation and the identification of modified amino acids in proteins.     

Applications of in-source fragmentation of protein ions for direct sequence analysis by delayed extraction MALDI-TOF mass spectrometry

In matrix-assisted laser desorption/ionization of proteins, there exists a certain amount of fast metastable decay immediately after laser irradiation. The fragment ions thus formed can be resolved and their m/z values measured accurately by employing delayed extraction linear time-of-flight mass spectrometry. At higher than threshold laser fluences, proteins exhibit a series of fragment ions providing useful sequence information. We also observe that when moderate amounts of salts are present in the sample with sinapinic acid being the matrix, the intensities of cn ions (N-terminal fragments) are enhanced compared to other types of fragment ions. This enhancement in cn ion signals allows direct sequencing of proteins. The cn ions are completely absent when Xxx-Pro bonds are encountered and are of lower intensity when Xxx-Gly bonds are involved. Further, the cn ion series is interrupted at Xxx-Cys, when the cysteine is involved in a disulfide bond. Upon reduction of the disulfide bonds, the series continues and information is available for longer stretches. Using 10-20 pmol of recombinant proteins, sometimes contiguous sequence information up to 70 residues is obtained in a matter of minutes. Applications of the technique to some recombinant proteins with intra- or interchain disulfide linkages are presented.     

Determination of an acetylcholinesterase inhibitor, MDL 73745, in dog plasma and urine by combined gas chromatography/negative ion chemical ionization mass spectrometry

A highly sensitive and specific assay has been developed for the determination of MDL 73745 [2,2,2-trifluoro-1-(3-trimethylsilyl-phenyl) ethanone] (I) and the internal standard (MDL 74398) at the nanomolar level in dog plasma and urine by gas chromatography/mass spectrometry. After a single-step extraction process, an aliquot was directly injected onto the gas chromatograph column. The mass spectrometer was run in the negative ion chemical ionization mode with ammonia as reagent gas, and was set to monitor the abundant M-. ion at m/z 246 of both compounds. The method yielded a linear response over the concentration range 0.1-10 pmol 100 microliters -1 plasma or urine. Within-day reproducibility at a concentration of 0.25, 1 and 5 pmol 100 microliters -1 plasma was 8.6%, 1.0% and 1.0%, respectively. The method was applied to the determination of I in plasma and urine after administration of 1 mg kg-1 i.v. and 10 mg kg-1 p.o. to dogs.