Mass spectrometric detection of fleeting neutral intermediates generated in electrochemical reactions

Towards the goal of on-line monitoring of transient neutral intermediates during electrochemical reactions, an electrochemistry-neutral reionization-mass spectrometry (EC-NR-MS) technique was developed in this work. The EC-NR setup consisted of a customized EC flow cell, a sonic spray ionization source, a heating tube, an ion deflector and an electrospray ionization source, which were respectively used for the precise control of the electrochemical reaction, solution nebulization, droplet desolvation, ion deflection and neutral intermediate ionization. Based on the EC-NR-MS approach, some long-sought neutral radicals including TPrA˙, DBAE˙ and TEOA˙, which belong to important reductive intermediates in electrochemiluminescence (ECL) reactions, were successfully identified which helps to clarify the previously unproven ECL reaction mechanism. These findings were also supported by spin-trapping experiments and the tandem MS technique. Accordingly, the EC-NR-MS method provides a direct solution for studying complicated electrochemical reactions, especially for detecting short-lived neutral radicals as well as ionic intermediates.

An electrochemistry-neutral reionization-mass spectrometry (EC-NR-MS) technique was developed for on-line studying the long-sought neutral radicals generated in electrochemical reactions.     

Electrochemiluminescent detection of labile radical intermediates of electrochemical reactions

The article considers the basic principles of electrochemiluminescent spin traps diagnostic method which consists of efficient trapping of intermediate labile radical products (free radicals of complex organic molecules) by electrogenerated radical ions of electrochemiluminescers in different liquid-phase systems including bioobjects. The method permits qualitative and quantitative identification of such particles by measurement of electrolysis radical recombination luminescence (electrogenerated chemiluminescence) intensity which is emitted by electrolyzed solutions of organic electrochemiluminescers. The unique possibilities of the method are illustrated on examples of experimental study of systems with sodium tetraphenylborate and chloride ion.     

Mass spectrometric interrogation of thioester-bound intermediates in the initial stages of epothilone biosynthesis

Direct detection of thioester intermediate mixtures bound to EpoC, a 195 kDa polyketide synthase, has been achieved using limited proteolysis and Fourier-transform mass spectrometry (FTMS). Incubation with various N-acetylcysteamine thioester (S-NAC) substrate mimics produced mass shifts on the EpoC ACP domain consistent with their condensation with an enzyme-bound carbanion produced by the decarboxylation of methylmalonyl-S-EpoC. Reconstitution of EpoA-ACP, EpoB, and EpoC gave a +165.0 Da mass shift consistent with the formation of the methylthiazolyl-methacrylyl product by incorporation of acetyl-CoA, cysteine, and methylmalonyl-CoA. Thioester-templated reaction intermediates and products are typically characterized by quantifying radioactive substrates, either enzyme bound or chemically hydrolyzed. In contrast, the MS-based methodology described here provides semiquantifiable ratios of free enzyme, intermediate, and product occupancy and reveals that certain substrates result in a >50% formation of nonproductive intermediates.     

Mass spectrometric detection of affinity purified crosslinked presented

Chemical crosslinking of proteins combined with mass spectrometric analysis of the tryptic digest of the products shows considerable promise as a tool for interrogating structure and geometry of proteins and protein complexes. An impediment to the use of this tool has been the difficulty of distinguishing crosslinked peptide pairs from non-crosslinked peptides, and from the products of side reactions. We describe the use of a commercially available biotinylated crosslinking reagent, sulfo-SBED, that allows affinity-based enrichment of crosslinked species. An intramolecular crosslink is prepared using the peptide neurotensin as a model system. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectra show the predicted crosslinking product, as well as several side products. Finally, we describe the optimized enrichment of biotinylated species, and reduction of non-specific binding, for a batch-mode affinity separation based on immobilized monomeric avidin.     

Mass spectrometric detection in chromatography. Trends and perspectives

The newest results in the use of miscellaneous mass spectrometric detection methods in various chromatographic techniques are compiled and critically evaluated. Examples of application in clinical chemistry, health care, and in the analysis of pharmaceuticals, environmental pollutants, foods and food products are given. The benefits and drawbacks of MS detection are discussed, and future trends are briefly discussed.     

Mass spectrometric detection of and similarities between 1-androgens

Regularly new anabolic steroids appear on the black market. In most cases these substances are marketed on websites or are confiscated during inspections. 1,(5alpha)-Androstene-17beta-ol-3-one, also known as 1-testosterone, is one of these substances presented to body-builders as a nutritional supplement or a pro-hormone. 1-Testosterone closely resembles the natural hormone testosterone except for a 1,2-double bound instead of a 4,5-double bound. 1-Androstene-3beta,17beta-diol is transformed into 1-testosterone after oral administration. 1-Testosterone, 1-androstene-3beta,17beta-diol and some other related 'new' anabolic steroids were studied with gas chromatography coupled to mass spectrometry (GC-MS) and Liquid chromatography coupled to tandem mass spectrometry (LC-MS2) methods. Similarities in spectra to known analytes, which may lead to pitfalls in the interpretation of the derivatised analytes, are discussed.     

Mechanisms of nucleophilic substitution reactions of methylated hydroxylamines with bis(2,4-dinitrophenyl)phosphate. Mass spectrometric identification of key intermediates

Mono- and dimethylation of hydroxylamine on nitrogen does not significantly affect rates of initial attack of NHMeOH and NMe(2)OH on bis(2,4-dinitrophenyl)phosphate (BDNPP), which is largely by oxygen phosphorylation. O-Methylation, however, blocks this reaction and NH(2)OMe then slowly reacts with BDNPP via N-attack at phosphorus and at the aryl group. With NHMeOH, the initial product of O-attack at phosphorus reacts further, either by reaction with a second NHMeOH or by a spontaneous shift of NHMe to the aryl group via a transient cyclic intermediate. There is a minor N-attack of NHMeOH on BDNPP in an S(N)2(Ar) reaction. Reactions occurring via N-attack are blocked by N-dimethylation, and reaction of NMe(2)OH with BDNPP occurs via O-attack, generating a long-lived product. Reaction mechanisms have been probed, and intermediates identified, by using both NMR and MS spectroscopy, with the novel interception of key reaction intermediates in the course of reaction by electrospray ionization mass and tandem mass spectrometry.     

Mass spectrometric intramolecular cyclization reactions of some 2-N-phenyliminoperhydro-1,3-oxazines

Mass spectrometric fragmentation of four monocyclic and eight condensed skeleton 2-N-phenyliminoper-hydro-1,3-oxazine derivatives have been characterized by metastable ion analysis, collision induced dissociation measurements, exact mass measurements and ion structural studies. Extensive rearrangement reactions, best characterized in terms of intramolecular cyclizations, took place. Namely, when R = H the N-cyclization was almost exclusive whereas in cases where R > H steric reasons rendered the O-cyclization more feasible. Part of the fragmentation of the R = H compounds seemed to occur via the amino form. On the other hand the methane chemical ionization spectra of cis- and trans-anellated 5,6-trimethylene-2-N-phenyliminoperhydro-1,3-oxazines proved that at least some imino structure was present in the gas phase.     

Mass spectrometric confirmation criterion for product-ion spectra generated in flow-injection analysis. Environmental application

The suitability of a confirmation criterion recently recommended in the Netherlands for gas chromatography with mass spectrometric detection (GC–MS), was evaluated for flow-injection analysis (FIA) with atmospheric pressure chemical ionisation MS–MS detection. The main feature of the criterion is that the relative ion abundances of the four diagnostic ions are taken into account. That is, for lower-intensity peaks, relative standard deviations may be higher; this is an advantage with chemical ionisation MS procedures. A series of triazines and their degradation products were used as test compounds. Tap and surface water samples spiked at 0.33 μg/l were analysed by means of a selected reaction monitoring MS–MS procedure. For all analytes but hydroxysimazine (3 transitions), 4–9 transitions could be selected which invariably met the demands of the criterion. Some of the transitions used originate from the ³⁷Cl isotopic mass of the parent compounds which provides additional structural information. Data for twenty surface water samples analysed by means of FIA–MS–MS as well as GC–MS and liquid chromatography with diode array UV and MS–MS detection gave essentially the same results over the 0.1–1.0 μg/l range. In two samples desethylatrazine was reported by FIA–MS–MS whereas this compound was not detected by GC–MS. For a first test, this is a promising result.     

Mass spectrometric detection of the Gibbs reaction for phenol analysis

This paper describes a new method for detecting phenols, by reaction with Gibbs reagent to form indophenols, followed by mass spectrometric detection. Unlike the standard Gibbs reaction, which uses a colorometric approach, the use of mass spectrometry allows for simultaneous detection of differently substituted phenols. The procedure is demonstrated to work for a large variety of phenols without para‐substitution. With para‐substituted phenols, Gibbs products are still often observed, but the specific product depends on the substituent. For para groups with high electronegativity, such as methoxy or halogens, the reaction proceeds by displacement of the substituent. For groups with lower electronegativity, such as amino or alkyl groups, Gibbs products are observed that retain the substituent, indicating that the reaction occurs at the ortho or meta position. In mixtures of phenols, the relative intensities of the Gibbs products are proportional to the relative concentrations, and concentrations as low as 1 μmol/L can be detected. The method is applied to the qualitative analysis of commercial liquid smoke, and it is found that hickory and mesquite flavors have significantly different phenolic composition.     

Mass spectrometric detection of siRNA in plasma samples for doping control purposes

Small interfering ribonucleic acid (siRNA) molecules can effect the expression of any gene by inducing the degradation of mRNA. Therefore, these molecules can be of interest for illicit performance enhancement in sports by affecting different metabolic pathways. An example of an efficient performance-enhancing gene knockdown is the myostatin gene that regulates muscle growth. This study was carried out to provide a tool for the mass spectrometric detection of modified and unmodified siRNA from plasma samples. The oligonucleotides are purified by centrifugal filtration and the use of an miRNA purification kit, followed by flow-injection analysis using an Exactive mass spectrometer to yield the accurate masses of the sense and antisense strands. Although chromatography and sensitive mass spectrometric analysis of oligonucleotides are still challenging, a method was developed and validated that has adequate sensitivity (limit of detection 0.25–1 nmol mL⁻¹) and performance (precision 11–21%, recovery 23–67%) for typical antisense oligonucleotides currently used in clinical studies.     

Mass spectrometric detection and identification of ortho, para-benzyltoluenes and oligotoluenes

Mass spectrometric detection and identification of ortho- and para-benzyltoluenes and oligotoluenes, C(21)-C(49), in several reaction mixtures was performed. Thus, the corresponding electron impact spectra were acquired and analyzed, this in addition to constant B/E linked scans and high-resolution data. Copyright 2000 John Wiley & Sons, Ltd.     

Thermodynamic and kinetic characteristics of intermediates of electrode reactions: Determination by direct and combined electrochemical methods

The potentialities of electrochemical and combined methods for the determination of thermodynamic characteristics and kinetic parameters of intermediates are analyzed. The electrochemical methods include polarography, versions of voltammetry, and chronopotentiometry. The combined methods combine nonelectrochemical (or indirect) generation of intermediates with electrochemical methods of their subsequent investigation: photomodulation voltammetry, method of polarography of pulsed-radiolysis products, method of indirect electrolysis, electrochemiluminescence, and a group of laser photoemission methods. Theoretical foundations and basic advantages and disadvantages of the laser photoemission methods (the measurement of photocurrents in potentiostatic conditions—method of time resolved voltammograms obtained under chopped illumination and the measurement of the kinetics of variations in the potential of an electrode after illuminating it by a pulsed laser—in coulostatic conditions) are described in detail. The potentialities of the laser photoemission methods for the determination of thermodynamic (standard potentials E ⁰ of redox pairs R/R^-, standard adsorption free energies) and kinetic (values of rate constants W ⁰ at an equilibrium potential, bulk lifetime of radicals Rand products of their reduction R^-) characteristics of intermediates are demonstrated by studying a trifluoromethyl radical.Translated from Elektrokhimiya, Vol. 41, No. 2, 2005, pp. 142–156.Original Russian Text Copyright © 2005 by Krivenko, Kotkin, Kurmaz.This revised version was published online in April 2005 with corrections to the article note and article title and cover date.     

Mass spectrometric detection of CP4 EPSPS in genetically modified soya and maize

The potential of protein fractionation hyphenated to mass spectrometry (MS) to detect and characterize the transgenic protein present in Roundup Ready soya and maize has been investigated. Genetically modified (GM) soya and maize contain the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Agrobacterium tumefaciens CP4, which confers resistance to the herbicide glyphosate. The GM soya and maize proteomes were fractionated by gel filtration, anion-exchange chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) prior to MS. This facilitated detection of a tryptic peptide map of CP4 EPSPS by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and nanoelectrospray ionization quadrupole time-of-flight (nanoESI-QTOF) MS. Subsequently, sequence information from the CP4 EPSPS tryptic peptides was obtained by nanoESI-QTOF MS/MS. The identification was accomplished in 0.9% GM soya seeds, which is the current EU threshold for food-labeling requirements.     

聚合物稳定的金簇合物上卤素吸附质：质谱检测及其对催化的影响

聚(N-乙烯基-2-吡咯烷酮)稳定的金簇合物(Au：PVP)的质谱结果表明,来源于合成前驱体的Cl吸附质主要存在于Au34和Au43簇合物上。金簇合物上Cl吸附质的数量不影响其催化有氧苯甲醇氧化性能,表明Cl原子与Au簇合物间存在较弱的键合作用。相反,用Br替代Au34和Au43簇合物上Cl显著抑制了其催化活性,但对其电子结构没有任何影响。这表明, Br原子与金簇合物的键合较强,在空间上堵塞了活性位。因Br吸附质而导致活性显著下降表明, Au34和Au43簇合物对Au：PVP催化活性起主要贡献。     

Liquid chromatography with post-column electrochemical treatment and mass spectrometric detection of non-polar compounds

The first hyphenation of high performance liquid chromatography (HPLC), electrochemical online oxidation and mass spectrometry (MS) is described. Ferrocenecarboxylic acid esters of various alcohols and phenols have been synthesized, separated by reversed-phase HPLC and oxidized (ionized) coulometrically prior to single quadrupole MS analysis using electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) interfaces. The dependence of the ionization on the electrochemical pretreatment is demonstrated. Limits of detection for selected derivatives range from 4 x 10(-9) to 4 x 10(-7) mol dm-3 depending on the individual compound and the selected interface.     

Radical intermediates generated in the reactions of l-arginine with hydroxyl radical and sulfate radical anion: A pulse radiolysis study

Reactions of l-arginine (Arg) with hydroxyl radical (OH) and sulfate radical anion (SO₄⁻) were kinetically investigated by the pulse radiolysis technique. Hydrogen abstraction from Arg by OH afforded redox chemically oxidizing, neutral, and reducing carbon-centered Arg radicals. Kinetic properties of the radicals indicated that the reducing species might include the δ-C-centered Arg radical and CO₂ radical anion. Similar transient spectra were observed in the SO₄⁻ reaction with Arg, suggesting direct oxidation at the guanidino group is less likely.