Detection of intermediates for the Eschweiler-Clarke reaction by liquid-phase reactive desorption electrospray ionization mass spectrometry

Desorption electrospray ionization mass spectrometry (DESI-MS) has been developed dramatically as a powerful tool for the rapid analysis of samples in their native environment. Here a novel application of DESI-MS was demonstrated for direct probing of the reactive intermediates in the liquid-phase Eschweiler-Clarke reaction, a reductive amination reaction whereby a primary (or secondary) amine is successively N-methylated using excess formaldehyde and formic acid. The intermediates ion species of sodiated amino alcohol ([I + Na](+)) and iminium ([II](+)), along with the corresponding protonated molecules of amine reactant ([M + H](+)) and end product ([III + H](+)), were simultaneously and unambiguously characterized by the positive ion DESI-MS in the native liquid-phase reactive condition. The operating variables were optimized for better analytical performance including the spray solvent composition (such as formic acid concentration, proportion of methanol-water), voltage applied, spray spatial distance and incident angle. The feasibility of the reactive DESI-MS detection of acid-formaldehyde methylations was further validated using amines of a large variety of chemical types (2 primary and 3 secondary amines). Thus, the liquid-phase reactive DESI-MS technique allows the direct analysis of reaction intermediates occurring in complex liquid solutions without sample preparation to provide a valuable insight into chemical reaction mechanisms.     

Biogenic aldehyde determination by reactive paper spray ionization mass spectrometry

Ionization of aliphatic and aromatic aldehydes is improved by performing simultaneous chemical derivatization using 4-aminophenol to produce charged iminium ions during paper spray ionization. Accelerated reactions occur in the microdroplets generated during the paper spray ionization event for the tested aldehydes (formaldehyde, n-pentanaldehyde, n-nonanaldehyde, n-decanaldehyde, n-dodecanaldehyde, benzaldehyde, m-anisaldehyde, and p-hydroxybenzaldehyde). Tandem mass spectrometric analysis of the iminium ions using collision-induced dissociation demonstrated that straight chain aldehydes give a characteristic fragment at m/z 122 (shown to correspond to protonated 4-(methyleneamino)phenol), while the aromatic aldehyde iminium ions fragment to give a characteristic product ion at m/z 120. These features allow straightforward identification of linear and aromatic aldehydes. Quantitative analysis of n-nonaldehyde using a benchtop mass spectrometer demonstrated a linear response over 3 orders of magnitude from 2.5 ng to 5 μg of aldehyde loaded on the filter paper emitter. The limit of detection was determined to be 2.2 ng for this aldehyde. The method had a precision of 22%, relative standard deviation. The experiment was also implemented using a portable ion trap mass spectrometer.     

Structural characterization of chelator-terminated dendrimers and their synthetic intermediates by mass spectrometry

Herein, we report the structural analysis of a novel family of iron-chelating dendrimers and their synthetic intermediates utilizing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and electrospray ionization ion trap (ESI IT) MS. These dendrimers share a benzene tricarbonyl core moiety attached to three tripodal branching units, each linking to three terminal groups, ranging from carboxyl, catechol and 3-hydroxy-6-methyl-pyran-4-one moieties and their protected analogs. In order to monitor the progression of dendrimer synthesis, all intermediates and final products were mass analyzed by conventional MALDI-TOF MS with and without alkali metal spiking. For structural characterization, interpretable post-source decay (PSD) and electrospray ionization ion trap MS/MS spectra were obtained from proton, sodium and potassium adducts of the dendrimers. One major route of dendrimer fragmentation was at or adjacent to the amide bonds either of the terminal chelating groups or near the core moiety. Fragmentation in the latter case was primarily at the N-terminal side of the amide bond and was directed by the proximity of a tertiary carbon of the branching unit. Furthermore, it was found that terminal ester, ether and amide linkages to the protecting and chelating groups could be sequentially broken in a single MS/MS spectrum through multiple cleavages resulting in product ions of decreasing intensity. Moreover, such cleavages could also be induced in a stepwise manner in a multistage ion trap MS(n) experiment.     

Structural characterization of reactive dyes using liquid secondary ion mass spectrometry/tandem mass spectrometry

Reactive Blue 19 (RB 19), its reactive form (RB 19-VS) and its hydrolyzed form (RB 19-OH) were examined using liquid secondary ion mass spectrometry/tandem mass spectrometry (LSIMS/MS/MS) in the negative-ion mode under low-energy collision conditions (240–300 eV). Structurally characteristic fragment ions were obtained, none of which has been previously reported for these reactive dyes. Among the ions obtained were SO₃^? ions, ions due to central amino cleavage and reactive group cleavage, and ions due to loss of SO₃ and SO₂. Possible pathways for the formation of product ions are proposed. The structural information obtained should help to characterize and identify reactive dyes better.     

Possible key intermediates in arsenic biochemistry: Synthesis and identification by liquid chromatography electrospray ionization mass spectrometry and high resolution mass spectrometry

Arsenic is a type 1 carcinogen and its toxicity is critically dependent on chemical speciation. However, after decades of research, the biogenesis of at least fifty naturally occurring arsenic species is still not well understood.Here, based on experimental work, it is proposed a set of pathways for the formation of multiple arsenic species that might help to clarify the present situation.These are focused on the thiol protein arsenic bond and on its interaction with reactive metabolites. In fact, arsenic bound to glutathione interacting with sulfur adenosyl methionine (SAM), MethylCB₁₂ and AdoCB₁₂, forms a number of complexes that might be key intermediates in arsenic biochemistry. These include dimethylarsino glutathione (DMAG) m/z 412 [M + H]⁺, synthesized non-enzymatically from glutathione and cacodylate. Trimethylarsonio glutathione (TMAG) m/z 426 [M]⁺ synthesized from DMA, GSH and SAM, apparently by a classical Challenger methylcarbonium attack. Tetramethyl arsonium ion m/z 135 [M]⁺ is formed in a third step, apparently by carbanion methylation. The presence of trimethylarsine oxide (TMAO) m/z 137 [M + H]⁺ is attributed to the hydrolysis of TMAG or TMA, or to carbanion methylation of dimethylarsinoyl glutathione (m/z 428 [M]⁺) formed from cacodylate and GSH. Cantoni type attacks of DMAG on SAM were unsuccessful, eventually due to competition of the trivalent S⁺ atom of SAM for the As^III atom attack. The presence of dimethylarsonio diglutathione (DMADG m/z 717 [M]⁺), is suggested to result from a GS^- attack on dimethylarsenoyl glutathione (m/z 428 [M + H]⁺). The presence of dimethylarsenoyladenosine (m/z 372 [M + H]⁺), trimethylarsenosugar adenine (m/z 370 [M]⁺), and dimethylthioarsenosugar adenine (m/z 388 [M + H]⁺), is explained by the synthesis of the pecursor dimethylarsonio-adenosine glutathione DMAAG (m/z 661 [M]⁺), a likely source of oxo-and trimethylated arsenosugars, as well as of thio-arsenosugars by the cleavage of its S-C bond. The results gathered suggest that cell vacuoles might play a major role in arsenic metabolism, and that the dominance of oxo-As sugars, in algae extracts, may be supported by a mechanism of synthesis independent of DMAAG (m/z 661).They also offer an explanation for the reason why arsenobetaine, and tetramethylarsonium are loosely bound to biotic tissues. Four arsenic species new to science, to the best of our knowledge, and a number of known arsenic compounds were synthesized in this work, identified by HPLC–ESI-MSⁿ and FTICR–ESI-MS, and suggestions regarding their mechanisms of synthesis were advanced. These results provide a framework for arsenic biochemistry which may explain the origin of a significant part of arsenic known metabolites.     

Identifying sites of protein modification by using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and on-line membrane preconcentration-capillary electrophoresis-tandem mass spectrometry

A strategy for rapidly identifying the number and sites of chemical or posttranslational modification of proteins is described. The use of matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry to determine the molecular weight of the adducted protein as well as map the proteolytic digest of peptides offers a rapid method to screen for the possible site of adduction. To unequivocally determine the amino acid sequence of the peptide bearing the adduct as well as structurally characteize the covalent modification, the peptide mixture is subjected to membrane preconcentration-capillary electrophoresis-mass spectrometry and tandem mass spectrometry (mPC-CE-MS/MS). The high resolving separation capability of capillary electrophoresis (CE) afford a chromatograhic step that lends itself to separation of complex mixtures of peptides with minimal sample loss. The membrane preconcentration-CE cartridge allows sample loading volumes 10,000-fold greater than conventional CE. In this work the binding site of the fluorescent label acrylodan to the intestinal fatty binding protein is characterized and shown to be covalently bound at lysine-27, by using mPC-CE-MS/MS.     

Analysis of triacetone triperoxide (TATP) and TATP synthetic intermediates by electrospray ionization mass spectrometry

The explosive triacetone triperoxide (TATP) has been analyzed by electrospray ionization mass spectrometry (ESI-MS) on a linear quadrupole instrument, giving a 62.5 ng limit of detection in full scan positive ion mode. In the ESI interface with no applied fragmentor voltage the m/z 245 [TATP + Na](+) ion was observed along with m/z 215 [TATP + Na - C(2)H(6)](+) and 81 [(CH(3))(2)CO + Na](+). When TATP was ionized by ESI with an applied fragmentor voltage of 75 V, ions at m/z 141 [C(4)H(6)O(4) + Na](+) and 172 [C(5)H(9)O(5) + Na](+) were also observed. When the precipitates formed in the synthesis of TATP were analyzed before the reaction was complete, a new series of ions was observed in which the ions were separated by 74 m/z units, with ions occurring at m/z 205, 279, 353, 427, 501, 575, 649 and 723. The series of evenly spaced ions is accounted for as oligomeric acetone carbonyl oxides terminated as hydroperoxides, [HOOC(CH(3))(2){OOC(CH(3))(2)}(n)OOH + Na](+) (n = 1, 2 ... 8). The ESI-MS spectra for this homologous series of oligoperoxides have previously been observed from the ozonolysis of tetramethylethylene at low temperatures. Precipitates from the incomplete reaction mixture, under an applied fragmentor voltage of 100 V in ESI, produced an additional ion observed at m/z 99 [C(2)H(4)O(3) + Na](+), and a set of ions separated by 74 m/z units occurring at m/z 173, 247, 321, 395, 469 and 543, proposed to correspond to [CH(3)CO{OOC(CH(3))(2)}(n)OOH + Na](+) (n = 1,2 ... 5). Support for the assigned structures was obtained through the analysis of both protiated and perdeuterated TATP samples.     

Kinetic intermediates in the folding of gaseous protein ions characterized by electron capture dissociation mass spectrometry

Alternative mechanisms propose that protein folding in solution proceeds either through specific obligate intermediates or by a multiplicity of routes in a "folding funnel". These questions are examined in the gas phase by using a new method that provides details of the noncovalent binding of solvent-free protein ions. Capture of an electron by a multiply charged cation causes immediate dissociation (ECD) of a backbone bond, but with negligible excitation of noncovalent bonds; thus ECD of a linear protein ion produces two measurable fragment ions only if these are not held together by noncovalent bonds. Thermal unfolding of 9+ ions of cytochrome c proceeds through the separate unfolding of up to 13 backbone regions (represented by 44 bond cleavages) with melting temperatures of <26 to 140 degrees C. An 0.25 s laser IR pulse induces unfolding of 9+ ions in <4 s in six of these regions, followed by their refolding in 2 min. However, for the 15+ ions a laser IR pulse causes slower unfolding through poorly defined intermediates that leads to far more ECD products (63% increase in bond cleavages) after 1 min, even more than heating to 140 degrees C, with refolding to a more compact conformation in 10 min. Random isomerization appears to produce a dynamic mixture of conformers that folds through a variety of pathways to the most stable conformer(s), consistent with a "folding funnel"; this might also be considered as an extension of the classical view to a system with a far smaller free energy change yielding multiple conformers. As cautions to inferring solution conformational structure from gas-phase data, no structural relationship between these gaseous folding intermediates and those in solution is apparent, consistent with reduced hydrophobic bonding and increased electrostatic repulsion. Further, equilibrium folding of gaseous ions can require minutes, and even momentary unfolding of an intermolecular complex during this time can be irreversible.     

Liquid chromatography and mass spectrometry of haem biosynthetic intermediates: a review

This article discusses the separation, analysis and characterisation of intermediates and oxidative by-products of the haem biosynthetic pathway by liquid chromatography and mass spectrometry. Techniques reviewed include high-performance liquid chromatography, ultra-high-performance liquid chromatography, capillary electrophoresis, ion mobility spectrometry, mass spectrometry and tandem mass spectrometry. The emphasis was on the analysis of biological and clinical samples.     

Handling considerations for the mass spectrometry of reactive organometallic compounds

Mass spectrometry is a powerful tool in disparate areas of chemistry, but its characteristic strength of sensitivity can be an Achilles heel when studying highly reactive organometallic compounds. A quantity of material suitable for mass spectrometric analysis often represents a tiny grain or a very dilute solution, and both are highly susceptible to decomposition due to ambient oxygen or moisture. This complexity can be frustrating to chemists and analysts alike: the former being unable to get spectra free of decomposition products and the latter often being poorly equipped to handle reactive samples. Fortunately, many creative solutions to such problems have been developed. This review summarizes some key methods for handling reactive samples in conjunction with the various ionization methods most frequently employed for their analysis.     

Trapping of reactive intermediates in dienone photochemistry by amines

Irradiation of 4-methyl-4-trichloromethyl-2,5-cyclohexadienones in amine solvents leads to the formation of the corresponding dichloromethyl cyclohexadienones by a process involving electron transfer from the amines to zwitterion intermediates.     

Rapid quantification of metabolic intermediates in blood by liquid chromatography-tandem mass spectrometry to investigate congenital lactic acidosis

A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been established to quantify metabolic intermediates, including lactate (Lac), pyruvate (Pyr), acetoacetate (ACAC) and 3-hydroxybutyrate (3-HB) in blood. Samples were deproteinized with methanol-acetonitrile solution, and analytes were separated on an adamantyl group-bonded reverse phase column and detected in multiple reaction monitoring mode. Total analysis time was 4 min per sample. Method validation results displayed that limits of quantification were 10.0 μmol L⁻¹ for Lac and Pyr, and 5.0 μmol L⁻¹for ACAC and 3-HB. The within- and between-run coefficients of variation were in the range of 1.2–6.4% for all analytes. The recoveries were ranged from 95.6 to 111.5%. The reference values of analytes were determined for the pediatric population. Duo to instability of Lac, Pyr and ACAC in vitro, a comprehensive stability assay was performed to determine optimal conditions for sample collection, pretreatment and storage. Results showed that precipitation of protein in blood at bedside combined with low storage temperature could effectively preserve the integrity of Lac, Pyr and 3-HB, but the precipitated protein accelerated degradation of ACAC. Isolation of supernatant fluid slowed degradation of ACAC. Supernatant samples could store at −20 °C for 10 days. The use of plasma or serum to determine these intermediates was not recommended. In this study, 450 samples from patients were analyzed, and 7 patients were diagnosed as congenital lactic acidosis. With the advantages of rapid, accurate and reliable, this method is very suitable for congenital lactic acidosis screening and researches related to energy metabolism.     

Analysis of natural products by tandem mass spectrometry employing reactive collisions with ethyl vinyl ether

Representative natural products from the diterpene dilactone, psorospermin and quabalactone classes were protonated, mass-selected and reacted with ethyl vinyl ether in a triple-quadrupole mass spectrometer. Minor differences in the structures of the compounds led to different reactivities toward the reagent, as indicated by the relative abundances of products such as the ethylated and vinylated compounds. Additional information is obtained from the dissociation products formed upon non-reactive, inelastic collisions with the neutral reagent. The daughter spectra have excellent signal: noise ratios and good reproducibility. The results demonstrate that the use of reactive collisions may supplement collision-activated dissociation in chemical analysis of large organic molecules by tandem mass spectrometry.     

液相色谱-二极管阵列检测/质谱联用在活性染料结构鉴定中的应用

基于多年运用液相色谱-二极管阵列检测/质谱(HPLC-DAD/MS)联用技术分析活性染料的实验积累,总结了HPLC-DAD/MS联用方法在商品活性染料结构鉴定中的重要应用。商品染料不需要纯化即可通过单次进样达到复杂组分的分离并得到光谱和质谱信息;结合衍生物质量差值法可推定染料的活性基种类和数目;采用二极管阵列检测器的全光谱扫描功能可以得到染料的发色体类别等有用的信息。HPLC-DAD/MS联用方法简便、快速、准确,尤其适用于多组分拼混的商品染料的结构鉴定,有较大的实用价值。通过对多组分商品染料活性灰、活性深黑、海洋蓝中关键组分的结构分析实例,介绍了HPLC-DAD/MS联用分析技术在未知商品活性染料结构分析中的应用。