Comparison of negative ion electrospray mass spectra measured by seven tandem mass analyzers towards library formation

A library of negative ion electrospray ionization mass spectra and tandem mass spectra (MS/MS) of sulfonated dyes has been developed for fast identification purposes. The uniform protocol has been elaborated and applied to the measurements of more than 50 anionic dyes. Three collision energies are selected in our protocol which ensures that at least one of them provides a suitable ratio of product ions to the precursor ion. The robustness is investigated with altered values of tuning parameters (e.g. the pressure of the nebulizing gas, the temperature and the flow rate of drying gas, and the mobile phase composition). The results of the inter-laboratory comparison of product ion mass spectra recorded on seven different tandem mass spectrometers (three ion traps, two triple quadrupoles and two hybrid quadrupole time of flight instruments) are presented for four representative anionic dyes--azo dye Acid Red 118, anthraquinone dye Acid Violet 43, triphenylmethane dye Acid Blue 1 and Al(III) metal-complex azo dye. The fragmentation patterns are almost identical for all tandem mass analyzers, only the ratios of product ions differ somewhat which confirms the possibility of spectra transfer among different mass analyzers with the goal of library formation.     

Mass spectrometric study of selected precursors and degradation products of chemical warfare agents

Selected precursors and degradation products of chemical warfare agents namely N,N-dialkylaminoethane-2-ols, N,N-dialkylaminoethyl-2-chlorides and some of related N-quaternary salts were studied by means of electrospray ionization-multiple tandem mass spectrometry (ESI-MS(n)). Proposed structures were confirmed with accurate mass measurement. General fragmentation patterns of these compounds are discussed in detail and suggested processes are confirmed using deuterated standards. The typical processes are elimination of alkene, hydrogen chloride, or water, respectively. Besides, elimination of ethene from propyl chain under specific conditions was observed and unambiguously confirmed using exact mass measurement and labelled standard. The potential of mass spectrometry to distinguish the positional isomers occurring among the studied compounds is reviewed in detail using two different MS instruments (i.e. ion trap and hybrid quadrupole-time of flight (Q-TOF) analyzer). A new microcolumn liquid chromatography (microLC)/MS(n) method was designed for the cases where the resolution based solely on differences in fragmentation is not sufficient. Low retention of the derivatives on reversed phase (RP) was overcome by using addition of less typical ion pairing agent (1 mM/l, 3,5-dinitrobenzoic acid) to the mobile phase (mixture water : acetonitrile).     

Preparation of 1,2-disubstituted-3-hydroxy-4(1H)-quinolinones and the influence of substitution on the course of cyclization

Synthesis of 1,2-disubstituted-3-hydroxy-4(1H)-quinolinones by the cyclization of N-substituted phenacyl or acetonyl anthranilates is described. Two methods were employed for cyclization of anthranilates. Heating in polyphosphoric acid has a wide scope of applicability. The thermal cyclization in boiling N-methylpyrrolidone is limited by steric effect.     

Determination of methylbenzoquate in premixes and animal feeds using liquid chromatography with fluorescence detection

A rapid analytical procedure was developed and tested for routine identification and quantification of methylbenzoquate in feeds by liquid chromatography (LC). The ground feed samples were extracted using methanol-water (80 + 20, v/v) at 65 degrees-70 degrees C in a water bath for premixes and in dichloromethane at 45 degrees C in a water bath for final feeds, respectively. The extract of final feeds was cleaned using solid-phase extraction on silica columns. Both the final feed and premix extracts were analyzed by reversed-phase LC on a NovaPak C18 column (3.9 x 150 mm; 4 microm) with methanol-acetonitrile-water-phosphoric acid (340 + 350 + 308 + 2, v/v) as mobile phase. Fluorescence detection was performed at excitation and emission wavelengths of 265 and 390 nm, respectively. Alternatively, post-column addition of sulfuric acid solution was used to decrease the determination limit. The recovery of methylbenzoquate, in a concentration range of 0.5-10 mg/kg, was 105.0 +/- 7.3%. The limit of quantitation, based on a signal-to-noise ratio of 10:1, was 48 microg/kg. The developed LC method was tested in an interlaboratory study. The interlaboratory repeatibility for both samples ranged from 7.1 to 10.6%; the interlaboratory reproducibility ranged from 11.7 to 15.2%. With the post-column addition of sulfuric acid, the limit of quantification was decreased by a factor of 50. Overall, the developed method is highly selective and can be used in routine analysis.     

Atmospheric pressure ionization mass spectrometry of purine and pyrimidine markers of inherited metabolic disorders

Purines and pyrimidines are of interest owing to their significance in processes in living organisms. Mass spectrometry is a promising analytical tool utilized in their analysis. Two atmospheric pressure ionization (API) methods (electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI)) in both negative and positive modes applied to selected purine and pyrimidine metabolites (markers of inherited metabolic disorders) were studied. APCI is less sensitive to alkali metal cations present in a sample and offers higher response than ESI for studied compounds. Both of the techniques afford quasi-molecular ions, but fragmentation also occurs to a certain extent. However, the application of collision-induced dissociation of quasi-molecular ions is essential to confirm a certain metabolite in a sample. Fragmentation of both positive and negative ions was evaluated using multi-stage mass spectrometric experiments. Typical neutral losses correspond to molecules NH(3), H(2)O, HCN, CO, H(2)NCN, HNCO and CO(2). The ion [NCO](-) arises in the negative mode. The cleavage of the glycosidic C-N bond is characteristic for relevant metabolites. Other neutral losses (CH(2)O, C(2)H(4)O(2) and C(3)H(6)O(3)) originate from fragmentation of the glycosidic part of the molecules. In addition to fragmentation, the formation of adducts of some ions with applied solvents (H(2)O, CH(3)OH) was observed. The composition of the solution infused into the ion source affects the appearance of the mass spectra. Tandem mass spectra allow one to distinguish compounds with the same molecular mass (uridine-pseudouridine and adenosine-2'-deoxyguanosine). Flow injection analysis APCI-MS/MS was tested on model samples of human urines corresponding to adenosine deaminase deficiency and xanthine oxidase deficiency. In both cases, the results showed potential diagnostic usefulness.     

Capillary electrophoresis/mass spectrometry: a promising tool for the control of some physiologically hazardous compounds. I-derivatives of 3-quinuclidinol

A method based on the coupling of capillary electrophoresis with mass spectrometry (CE/MS) was developed for the monitoring of 3-quinuclidinol and its four N-alkyl derivatives (methyl, ethyl, propyl and isopropyl derivatives). A fragmentation study (collision-induced dissociation of ions in an ion trap) and optimization of the ion optics set-up for CE/MS experiments using direct infusion of a methanolic solution of the standards into the mass spectrometer were carried out in advance. Molecular ions of all quaternary compounds and the quasi-molecular ion [M + H]+ of free 3-quinuclidinol prevail in the mass spectra. In the MS/MS of propyl and isopropyl derivatives, the elimination of the alkyl chain dominates, leading to the ion at m/z 128. The fragmentation of the other compounds is more complex. Previous CE separation of the mixture of isobaric propyl and isopropyl derivatives is necessary for their unambiguous identification. A 10 mM ammonium acetate buffer (pH 4.0) is the optimum running electrolyte, allowing the CE separation of methyl, ethyl, propyl and isopropyl derivatives. A 0.5% (v/v) solution of acetic acid in methanol provides sufficient detection sensitivity when used as the sheath liquid. Limits of detection of 0.1 ppm for 3-quinuclidinol and 0.05 ppm for quaternary derivatives were achieved under the optimum conditions. The optimized method was applied to the determination of 3-quinuclidinol and related quaternary derivatives spiked into a sample of pond water. The experimental set-up for CE/MS/MS was investigated, which strongly increases the identification capability of the technique.     

Signal enhancement in desorption nanoelectrospray ionization by custom‐made inlet with pressure regulation

The efficiency of desorption/ionization becomes more critical as the sampled surface area decreases. Desorption electrospray and desorption nanoelectrospray belong to ambient ionizations and enable direct surface analysis including mass spectrometric imaging. Lateral resolution in tens of micrometers was demonstrated for desorption nanoelectrospray previously, but sensitivity of the surface scan can be an issue. For desorption electrospray, the drag force in the source is driven by the flow of used gases and vacuum suction. Ion signal intensity can be improved by controlling the nebulizing gas flow rate or auxiliary pumping of a closed compartment in front of the mass spectrometer inlet. Because nanoelectrospray generates charged droplets without the assistance of a nebulizing gas, only vacuum suction drives the gas flow. In this study, the effect of pressure drop between the atmospheric and evacuated region of a mass spectrometer on the ion signal intensity was investigated for desorption nanoelectrospray. A modification of the commercial inlet was designed. An auxiliary pump was directly connected to an inner compartment of the modified mass spectrometer inlet through a needle valve that enabled the regulation of the reduced pressure. Adjustment of the pressure drop significantly increased signal intensity (more than one order of magnitude in some cases). To a lesser extent, the temperature of a heated capillary (an integral part of the inlet) also influenced the signal intensity. The applicability of desorption nanoelectrospray equipped with pressure regulation was demonstrated by the analysis of synthetic cathinones or a pill of paracetamol. Because pressure in the inlet depends on the diameters of orifices and the power of vacuum systems of mass spectrometers, the effect of the pressure regulation can be different for different instruments. Nevertheless, the presented results confirmed the importance of pressure drop‐driven transport for desorption nanoelectrospray efficiency and can encourage its new applications.     

Continuous flow-extractive desorption electrospray ionization: Analysis from “non-electrospray ionization-friendly” solvents and related mechanism

Due to their low polarities and dielectric constants, analytes in solvents such as hexane, chloroform, and ethyl acetate exhibit poor electrospray ionization (ESI) efficiency. These are deemed to be “non-ESI-friendly” solvents. Continuous flow extractive desorption electrospray ionization (CF-EDESI) is a novel ambient ionization technique that was recently developed in our group to manipulate protein charge distributions. Here we demonstrate its potential for ionizing analytes from non-ESI-friendly solvents. This feature makes CF-EDESI attractive to the general analytical community due to its apparent potential in lipidomics, normal phase separations, and hyphenation of mass spectrometry with HPLC-NMR systems. In this context, interest was subsequently initiated to discern mechanistic aspects of CF-EDESI. To achieve this, mechanistic experiments associated with a seemingly similar ambient ionization technique, extractive electrospray ionization (EESI), were emulated to compare CF-EDESI and EESI. Analysis of a series of fatty acids in multiple solvents in the negative ionization mode revealed differences between the two techniques. Whereas EESI has been previously shown to operate via extraction of analytes into the spray solvent, data presented here for CF-EDESI point toward a liquid-liquid mixing process to facilitate ionization. Further, a partial factorial design experiment was performed to evaluate the effects of different experimental variables on signal intensity. Sample flow rate was confirmed to be among the most significant factors to affect sensitivity. As a whole, the work presented provides greater insight into a new ambient ionization process, which exhibits expanded capabilities over conventional ESI; in this case, for direct analysis from non-ESI-friendly solvents.     

Study of electrochemical oxidation of cyanidin glycosides by online combination of electrochemistry with electrospray ionization tandem mass spectrometry

Abstract  

Electrochemistry coupled with mass spectrometry (EC–MS) is a promising analytical tool for the online study of oxidation processes of anthocyanins. Two cyanidin glycosides, cyanidin-3-galactoside (ideain) and cyanidin-3,5-diglucoside (cyanin), were subjected to electrochemical oxidation and subsequent online mass spectrometric identification of the formed products. Application of relatively low working potentials (around 100 mV vs. Pd/H₂) using a porous graphite coulometric electrode yielded detectable oxidation products. As determined by hydrodynamic voltammetry, the monoglycosylated analogue undergoes anodic oxidation easier than the diglycosylated one. As a first step of the electrochemically induced oxidation, incorporation of a hydroxyl group was observed for both glycosides. Besides, an oxidative condensation of two anthocyanin molecules was observed. The proposed oxidative condensation was further confirmed by consecutive fragmentation (i.e., collection of MS², MS³, and MS⁴ spectra) in which corresponding subsequent losses of the sugar moiety were observed.