Organic Mass Spectrometry Research Articles

High‐precision isotopic analysis of palmitoylcarnitine by liquid chromatography/electrospray ionization ion‐trap tandem mass spectrometry

Single quadrupole gas chromatography/mass spectrometry (GC/MS) has been widely used for isotopic analysis in metabolic investigations using stable isotopes as tracers. However, its inherent shortcomings prohibit it from broader use, including low isotopic precision and the need for chemical derivatization of the analyte. In order to improve isotopic detection power, liquid chromatography/electrospray ionization ion-trap tandem mass spectrometry (LC/ESI-itMS2) has been evaluated for its isotopic precision and chemical sensitivity for the analysis of [13C]palmitoylcarnitine. Over the enrichment range of 0.4-10 MPE (molar % excess), the isotopic response of LC/ESI-itMS2 to [13C]palmitoylcarnitine was linear (r = 1.00) and the average isotopic precision (standard deviation, SD) was 0.11 MPE with an average coefficient of variation (CV) of 5.6%. At the lower end of isotopic enrichments (0.4-0.9 MPE), the isotopic precision was 0.05 MPE (CV = 8%). Routine analysis of rat skeletal muscle [13C4]palmitoylcarnitine demonstrated an isotopic precision of 0.03 MPE for gastrocnemius (n = 16) and of 0.02 MPE for tibialis anterior (n = 16). The high precision enabled the detection of a small (0.08 MPE) but significant (P = 0.01) difference in [13C4]palmitoylcarnitine enrichments between the two muscles, 0.51 MPE (CV = 5.8%) and 0.43 MPE (CV = 4.6%), respectively. Therefore, the system demonstrated an isotopic lower detection limit (LDL) of < or =0.1 MPE (2 x SD) that has been impossible previously with other organic mass spectrometry instruments. LC/ESI-itMS2 systems have the potential to advance metabolic investigations using stable isotopes to a new level by significantly increasing the isotopic solving power.

Ion-mobility study of two functionalized pentacene structural isomers using a modified electrospray/triple quadrupole mass spectrometer

We report ion-mobility measurements with a modified triple quadrupole mass spectrometer fitted with an ion molecule reactor (IMR) designed to investigate ion molecule reactivity in organic mass spectrometry. Functionalized pentacene ions, which are generally unreactive were chosen for study to decouple drift/diffusion effects from reactivity (including clustering). The IMR is equipped with a variable axial electrostatic drift field (ADF) and is able to trap ions. These capabilities were successfully employed in the measurement of ion mobilities in different modes of IMR operation. Theoretical modeling of the drift dynamics and the special localization of the large ion packet was successfully implemented. The contribution of the quadrupole RF field to the drift dynamics also was taken into consideration.

Development and validation of a liquid chromatography–tandem mass spectrometry method for the separation of conjugated and unconjugated 17α- and 17β-boldenone in urine sample

Natural occurrence or illegal treatment of boldenone (BOLD) presence in cattle urine is under debate within the European Union. Separation of conjugated and unconjugated forms of 17α-boldenone (α-BOLD) and 17β-boldenone (β-BOLD) and presence of related molecules as androsta-1,4-diene-3,17-dione (ADD) appear critical points for the decision of an illegal use. The aim of this study is a new analytical approach of BOLD and ADD confirmation in cattle urine. The separation between conjugated and unconjugated forms of BOLD was obtained by a preliminary urine liquid–liquid extraction step with ethyl acetate. In this step the organic phase extracts only unconjugated BOLD and ADD, while BOLD in conjugated form remain in urine phase. Afterwards the urine phase, contains conjugated BOLD, was subjected to an enzymatic deconjugation. Solid-phase extraction (OASIS-HLB Waters) was used for the purification and concentration of analytes in organic and urine phases and liquid chromatography ion electrospray tandem mass spectrometry (LC–MS–MS) was applied for the confirmation of BOLD and ADD, using deuterium-labelled 17β-boldenone (BOLD-d3) as internal standard. The method was validated as a quantitative confirmatory method according to the Commission Decision 2002/657/CE. The results obtained demonstrate that the developed method show very high specificity, precision, trueness and ruggedness. Decision limits (CCα) smaller than 0.5ngmL−1 were obtained for each analyte.

Formation of C 7H 7+ ions from ethylbenzene and o-xylene ions: Fragmentation versus isomerization

The C 7H 7 + ion, existing in three major isomeric forms (tropylium, benzylium and tolylium), is one of the most fascinating species in organic mass spectrometry. Yet the properties of the three isomers are still under some debate. In a recent experiment we have measured the rate constants for the formation of C 7H 7 + from energy-selected ethylbenzene and o-xylene ions [M. Malow, M. Penno, K.M. Weitzel, J. Phys. Chem. A, 107, (2003) 10625]. In the current work we present an extended kinetic analysis taking into account competition of isomerization and fragmentation in both ethylbenzene and o-xylene ions. The comparison of the kinetic model with experimental data indicates that isomerization of the precursors to a common intermediate (the methyl-cycloheptatriene ion) prior to fragmentation does not play an important role in the threshold region. Most likely the effective barriers for isomerization of the precursors are significantly higher than the thermochemical onset of fragmentation.

Organic mass spectrometry in archaeology: evidence for Brassicaceae seed oil in Egyptian ceramic lamps

An analytical procedure based on alkaline hydrolysis and silylation followed by GC/MS analysis was employed to study the formation of characteristic acidic compounds and the development of a distinctive chromatographic pattern in the course of accelerated ageing tests on Brassicaceae seed oil. On the basis of mass spectra of trimethylsilyl derivatives, the main degradation products were identified as alpha,omega-dicarboxylic, omega-hydroxycarboxylic and dihydroxycarboxylic acids, including 11,12-dihydroxyeicosanoic acid and 13,14-dihydroxydocosanoic acid. The mass spectra of both these compounds are characterised by fragment ions arising from the alpha cleavage of the bond between the two vicinal trimethylsiloxy groups, resulting in fragments at m/z 215 and 345 for 11,12-dihydroxyeicosanoic acid, and at m/z 215 and 373 for 13,14-dihydroxydocosanoic acid. Other significant fragment ion-radicals from rearrangement process at m/z [M - 90](+*), [M - 142](+*), 204 as well as fragment ions at m/z [M - 15](+), [M - 105](+), 217 are present in the mass spectra of both the compounds. The results obtained for reference materials were compared with those relating to archaeological organic materials recovered in Egyptian pottery lamps. The occurrence of the same characteristic degradation products found in the reference materials subjected to accelerated ageing indicates an unambiguous origin for the organic archaeological remains and represents the chemical evidence for the use of oil from seeds of Brassicaceae as illuminant.

Potential for Using Isotopically Altered Metalloproteins in Species-Specific Isotope Dilution Analysis of Proteins by HPLC Coupled to Inductively Coupled Plasma Mass Spectrometry

The production and evaluation of an isotopically enriched metalloprotein standard for use as a calibrant in species-specific isotope dilution analysis by HPLC coupled to inductively coupled plasma mass spectrometry is described. Using a model system involving the copper-containing protein rusticyanin (Rc) from the bacterium Acido-thiobacillus ferrooxidans, it was possible to demonstrate the analytical conditions that could be used for the measurement of metalloproteins by on-line IDMS analysis. Rc was chosen because it is a well-characterized protein with an established amino acid sequence and can be produced in suitable quantities using a bacterial recombinant system. Three different forms of the protein were studied by organic and inorganic mass spectrometry: the native form of the protein containing a natural isotopic profile for copper, an isotopically enriched species containing virtually all of its copper as the 65Cu isotope, and the nonmetalated apo form. Incorporation of the copper isotopes into the apo form of the protein was determined using a UV-vis spectrophotometric assay and shown to be complete for each of the copper-containing species. The experimental conditions required to maintain the conformational form of the protein with a nonexchangeable copper center were established using +ve electrospray mass spectrometry. A pH 7.0 buffer was found to afford the most appropriate conditions, and this was then used with HPLC-ICP-MS to verify the stability of the copper center by analysis of mixtures of different isotopic solutions. No exchange of the enriched copper isotope from Rc with an added naturally abundant inorganic copper cation was observed under a neutral pH environment, indicating that species-specific ID-MS analysis of metalloproteins is possible.

Metal Salts for Molecular Ion Yield Enhancement in Organic Secondary Ion Mass Spectrometry: A Critical Assessment

In a search for molecular ion signal enhancement in organic SIMS, the efficiency of a series of organic and inorganic salts for molecular cationization has been tested using a panel of nonvolatile molecules with very different chemical characteristics (leucine enkephalin, Irganox 1010, tetraphenylnaphthalene, polystyrene). The compounds used for cationization include alkali bromide and group Ib metal salts (XBr with X = Li, Na, K; CF3CO2Ag; AgNO3; [CH3COCH=C(O-)CH3]2Cu; AuCl3). Alkali ions, very good for polar molecule cationization, prove to be of limited interest for nonpolar molecules such as polystyrene. Silver trifluoroacetate displays excellent results for all the considered molecules, except for leucine enkephalin (which might be due to the use of different solvents for the analyte and the salt). Instead, silver nitrate mixed with leucine enkephalin in an ethanol solution provides intense molecular signals. The influence of the respective concentrations of analyte and salt in solution, of the silver trifluoroacetate solution stability, and of the sample microstructure on the secondary ion intensities are also investigated. The results of other combinations of analyte and salts are reported. Finally, the use of salts is critically compared to other sample preparation procedures previously proposed for SIMS analysis of large organic molecules.

Melatonin prevents the free radical and MADD metabolic profiles induced by antituberculosis drugs in an animal model

The objective was to determine the effect of combined antituberculosis (anti-TB) drug therapy and an antioxidant, melatonin, on the free radical and organic acid profiles in an experimental rat model. A combined anti-TB drug, Rifater, consisting of 12.0 mg rifampicin, 0.8 mg isoniazid, and 23.0 mg pyrazinamide and 18.56 microg melatonin/kg body weight per day (corresponding to average physiological human intake) were orally administered to Sprague-Dawley rats. Hydroxyl radical production was monitored by quantifying 2,3-dihydroxybenzoic acid produced after intraperitonial sodium salicylate injections. Organic acid extractions and gas chromatography-coupled mass spectrometry analyses were performed on collected urine samples. The results show hydroxyl radicals (P = 0.0019) and organic acids (P-value range: 0.037 to <0.001), characteristic of a multiple acyl-CoA dehydrogenase defect (MADD), were elevated with Rifater treatment and these elevations were significantly lowered with melatonin pretreatment (P-value range: 0.031 to <0.001), probably because of its inherent antioxidant activity. We conclude that hydroxyl radical production and an increased organic acid profile induced by anti-TB medication indicates inhibition of the electron transport chain. We also conclude that free radicals leading to clinical symptoms associated with an MADD metabolic profile induced by anti-TB treatment could be alleviated by melatonin intervention.

Interest of silver and gold metallization for molecular SIMS and SIMS imaging

The metallization procedure, recently proposed for signal improvement in organic secondary ion mass spectrometry (metal assisted-SIMS or MetA-SIMS), has been quantitatively evaluated for Irganox 1010 and polystyrene fragment and quasimolecular ions. In addition to gold, we investigate the effect of silver evaporation as a sample treatment prior to static SIMS analysis. Ion yields and yield enhancement factors are compared for Ag and Au-metallized molecular films, pristine coatings on silicon and sub-monolayers of the same molecules adsorbed on silver and gold. The results are sample-dependent but, as an example, the yield enhancement calculated for metallized polymer additive (Irganox 1010) films with respect to untreated coatings is larger than two orders of magnitude for the quasimolecular ion and a factor between 1 and 10 for characteristic fragments. The interest of the method for imaging SIMS applications is illustrated by the study of a non-uniform coating of polystyrene oligomers on a 100 μm polypropylene film.

Interest of silver and gold metallization for molecular SIMS and SIMS imaging

The metallization procedure, recently proposed for signal improvement in organic secondary ion mass spectrometry (metal assisted-SIMS or MetA-SIMS), has been quantitatively evaluated for Irganox 1010 and polystyrene fragment and quasimolecular ions. In addition to gold, we investigate the effect of silver evaporation as a sample treatment prior to static SIMS analysis. Ion yields and yield enhancement factors are compared for Ag and Au-metallized molecular films, pristine coatings on silicon and sub-monolayers of the same molecules adsorbed on silver and gold. The results are sample-dependent but, as an example, the yield enhancement calculated for metallized polymer additive (Irganox 1010) films with respect to untreated coatings is larger than two orders of magnitude for the quasimolecular ion and a factor between 1 and 10 for characteristic fragments. The interest of the method for imaging SIMS applications is illustrated by the study of a non-uniform coating of polystyrene oligomers on a 100 μm polypropylene film.

14 Organic gas-phase ion chemistry

This review summarizes the physical organic chemistry studies utilizing gas-phase ion chemistry in the past year. For more than 20 years, gas-phase ion chemistry has provided insight into mechanisms of chemical reactions through studies of reactivity, structure, and thermochemistry of neutral and ionic substrates. Whereas initial studies often involved the use of simple mass spectrometric methods (by today's standards) of an ion source and a detector, modern methods are more sophisticated, as are the problems being investigated. Thus, in addition to gaining insight into the role that solvation plays in stabilizing ionic structures, as with Kass and Wang's studies of zwitterionic pyridinium carboxylates, or the effect of ionization on reaction potential energy surfaces, as illustrated by the electron-catalyzed Cope rearrangement study by Hammad and Wenthold, gas-phase ion chemistry has been applied to modern, burgeoning areas of chemistry including host–guest chemistry, in the report of “molecular mousetraps” by Beauchamp and co-workers, and abiogenesis, where Cooks and co-workers tout the potential role of ionic serine clusters.As with last year, we identified papers for this review by considering contributions to the standard organic, physical, and mass spectrometry journals. The selected articles are those that address the issue of reactivity and structure of organic molecules. Because our emphasis in this work is on the chemistry that is observed and the main conclusions, information regarding the instrumental technique is generally not provided. Most reactivity studies were carried out by using Fourier transform-ion cyclotron resonance (FT-ICR), quadrupole ion trap, or flowing afterglow techniques, but some studies involved the use of tandem methods. Most of the spectroscopic studies involved the use of photoelectron spectroscopy, or a variation thereof. Even more so than we did last year, we have omitted most papers that are mainly organometallic or theoretical in nature. Yet, despite the more stringent selection, we have included 45 more papers than we did last year.In last year's article, we noted the problem of whether to include biological studies. As with most areas of organic chemistry, the biological influence in gas-phase ion chemistry is undeniable, and evident in all aspects of this review. This year we also include a section on biological studies, which included studies of larger substrates such as peptides.The articles summarized in this review are organized into broad classifications: reactivity, reactive species, spectroscopy, and thermochemistry. As has been traditionally the case with this report, we separate the reactivity studies into multiple sections including one specially addressing stereoselective reactivity and stereochemical analysis, and one addressing structures and energetics of biologically relevant molecules.A 2003 edition of Topics in Current Chemistry had a mass spectrometry focus, with reviews covering aspects of reactivity, metalorganic chemistry and methodology. Many of the topics included in the volume were related to organic mass spectrometry.

Collisional dissociation in plasma source mass spectrometry: A potential alternative to chemical reactions for isobar removal

There has been a great deal of interest over the last half-dozen years in the use of selective chemical reactions to remove problematic species from ion beams extracted from analytical plasma sources. While these chemistries are well founded in principle, they require a priori knowledge of spectral contributors and all concomitant sample species. Different chemistries are required on both the analyte and matrix-specific bases. This approach has led to the generation of instrument/analysis cookbooks very much akin to those developed for graphite furnace atomization atomic absorption spectrophotometry (GFA-AAS). In addition to chemical reactions, the organic mass spectrometry community has employed kinematic (collisional) methods to dissociate molecular ions where the production of stable fragments is achieved to yield structurally significant product spectra. In this same way, collision-induced dissociation (CID) has been employed to remove molecular isobars in inductively-coupled plasma (ICP) and glow discharge (GD) mass spectrometries. Discussed here are the results of those studies, dating back over a dozen years ago, and how they compare in practical terms with the current commercial implementation of chemical reactions. Interestingly, there have been no publications that have actually compared the use of chemical and kinematic methods on the same instrumentation to achieve selective and universal analysis schemes. This “perspectives” paper seeks to present those previous CID works to a plasma source mass spectrometry community, wherein multi-quadrupole systems are becoming commonplace, in hopes of evoking substantial experimental work and scientific debate that may pay off practical benefits to the general user.

Organic secondary ion mass spectrometry: sensitivity enhancement by gold deposition.

Hydrocarbon oligomers, high-molecular-weight polymers, and polymer additives have been covered with 2-60 nmol of gold/cm2 in order to enhance the ionization efficiency for static secondary ion mass spectrometry (s-SIMS) measurements. Au-cationized molecules (up to -3,000 Da) and fragments (up to the trimer) are observed in the positive mass spectra of metallized polystyrene (PS) oligomer films. Beyond 3,000 Da, the entanglement of polymer chains prevents the ejection of intact molecules from a "thick" organic film. This mass limit can be overcome by embedding the polymer chains in a low-molecular-weight matix. The diffusion of organic molecules over the metal surfaces is also demonstrated for short PS oligomers. In the case of high-molecular-weight polymers (polyethylene, polypropylene, PS) and polymer additives (Irganox 1010, Irgafos 168), the metallization procedure induces a dramatic increase of the fingerprint fragment ion yields as well as the formation of new Aucationized species that can be used for chemical diagnostics. In comparison with the deposition of submonolayers of organic molecules on metallic surfaces, metal evaporation onto organic samples provides a comparable sensitivity enhancement. The distinct advantage of the metal evaporation procedure is that it can be used for any kind of organic sample, irrespective of thickness, opening new perspectives for "real world" sample analysis and chemical imaging by s-SIMS.

Integral membrane proteins of the chloroplast envelope: identification and subcellular localization of new transporters.

A two-membrane system, or envelope, surrounds plastids. Because of the integration of chloroplast metabolism within the plant cell, the envelope is the site of many specific transport activities. However, only a few proteins involved in the processes of transport across the chloroplast envelope have been identified already at the molecular level. To discover new envelope transporters, we developed a subcellular proteomic approach, which is aimed to identify the most hydrophobic envelope proteins. This strategy combined the use of highly purified and characterized membrane fractions, extraction of the hydrophobic proteins with organic solvents, SDS/PAGE separation, and tandem mass spectrometry analysis. To process the large amount of MS/MS data, a blast-based program was developed for searching in protein, expressed sequence tag, and genomic plant databases. Among the 54 identified proteins, 27 were new envelope proteins, with most of them bearing multiple alpha-helical transmembrane regions and being very likely envelope transporters. The present proteomic study also allowed us to identify common features among the known and newly identified putative envelope inner membrane transporters. These features were used to mine the complete Arabidopsis genome and allowed us to establish a virtual plastid envelope integral protein database. Altogether, both proteomic and in silico approaches identified more than 50 candidates for the as yet previously uncharacterized plastid envelope transporters. The predictable function of some of these proteins opens up areas of investigation that may lead to a better understanding of the chloroplast metabolism. The present subcellular proteomic approach is amenable to the analysis of the hydrophobic core of other intracellular membrane systems.

Estuarine/marine UDOM as characterized by size-exclusion chromatography and organic mass spectrometry

High performance size-exclusion chromatography (HPSEC) and direct temperature-resolved mass spectrometry (DT-MS) were used to explore molecular weight distributions and molecular level characteristics within marine/estuarine ultrafiltered dissolved organic matter (UDOM, >1 kDa) collected from the lower Chesapeake Bay, USA and the Oosterschelde estuary, The Netherlands. Initial HPSEC characterization indicates that the overall size distribution is similar in all the UDOM samples; however, there are distinct variations among the samples, especially in the low molecular weight region. As a preliminary study of molecular level variations with apparent molecular size, the Oosterschelde UDOM sample was separated into molecular weight fractions using HPSEC. These fractions were then analyzed by DT-MS. The size fractions within the UDOM sample yielded distinctly different mass spectra; the larger size classes were enriched in aminosugars, deoxysugars, and methylated sugars while the smaller size classes were enriched in hexose sugars. There is also evidence that hexose sugars appear in at least two structures with highly different molecular weights and potentially highly different source and sink functions within the marine water column.

The potential for elemental analysis in biotechnology

The ICP-MS brings particular attributes to the method that distinguishes it from organic mass spectrometry: relative insensitivity to concomitant organic or metallic species, equivalent sensitivity to all similarly-tagged complexes, inherent ability to provide absolute quantitation and linearity of response. All these positive features fit the requirements of modern-day biotechnology. In this work, the potential of ICP-MS is discussed in the context of several urgent problems of biotechnology including: the determination of the state of phosphorylation of proteins, quality control of biologically active materials and control of efficiency of cell line transfections.

Sensitive determination of pethidine in body fluids by surface ionization organic mass spectrometry

We have presented a simple and sensitive method for determining pethidine, a narcotic analgesic drug in body fluids by gas chromatography (GC)/surface ionization organic mass spectrometry (SIOMS). Good linearity was obtained in the range of 0.625–25 ng/ml of whole blood and urine by mass chromatography, and in the range of 0.05–2 ng/ml of whole blood by selected ion monitoring (SIM). Pethidine and diphenylpyraline (internal standard) were extracted from body fluids with Bond Elut Certify cartridges; their recoveries were above 95%. The detection limits (signal-to-noise ratio=3) were estimated to be 0.2 ng/ml of whole blood or urine by mass chromatography, 0.02 ng/ml of whole blood by SIM.

Effectiveness of atomic and polyatomic primary ions for organic secondary ion mass spectrometry

With the increased application of polyatomic primary ions for organic secondary ion mass spectrometry, a method to compare the performance of the various available projectiles is needed. In this work, primary ion performance was evaluated based on analyte-specific secondary ion yield, the amount of fragmentation, and the amount of metastable decay induced by each primary ion. These measurements were used to construct a projectile efficiency number for each primary ion applied to a specific analyte. An organic acid, phospholipids, and alkyl sulfates, prepared as multilayer and monolayer surfaces were analyzed with various atomic and polyatomic primary ions. It was found that polyatomic primary ions are best suited for multilayer organic samples whereas atomic primary ions are most effective for monolayer organic samples. Results are compared to other studies that measure the ion formation efficiency of various primary ions.

Organic ion imaging of biological tissue with secondary ion mass spectrometry and matrix-assisted laser desorption/ionization.

Organic secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry can be used to produce molecular images of samples. This is achieved through ionization from a clearly identified point on a flat sample, and performing a raster of the sample by moving the point of ionization over the sample surface. The unique analytical capabilities of mass spectrometry for mapping a variety of biological samples at the tissue level are discussed. SIMS provides information on the spatial distribution of the elements and low molecular mass compounds as well as molecular structures on these compounds, while MALDI yields spatial information about higher molecular mass compounds, including their distributions in tissues at very low levels, as well as information on the molecular structures of these compounds. Application of these methods to analytical problems requires appropriate instrumentation, sample preparation methodology, and a data presentation usually in a three-coordinate plot where x and y are physical dimensions of the sample and z is the signal amplitude. The use of imaging mass spectrometry is illustrated with several biological systems.

Situations of poisoning and analytical toxicology in Japan

Unprecedented poisoning terrorism by use of sarin took place in Japan in 1994 and 1995. On July 25, 1998, a curry poisoning incident in Wakayama occurred resulting in the death of four people and injury of 63 people. Since then, more than 30 imitative poisoning cases have been reported by mass communication within 1 year. In this brief review, we present some details of a series of poisoning cases and measures taken by the Japanese Government and non-government groups. In addition some research projects on analytical toxicology being conducted in our laboratories are also introduced; gas chromatography (GC) with cryogenic oven trapping for volatile organic compounds, and GC/surface ionization organic mass spectrometry for tertiary amino compounds are described especially as new techniques for biological samples.