An experimental and computational investigation on the fragmentation behavior of enaminones in electrospray ionization mass spectrometry

The dissociation pathways of protonated enaminones with different substituents were investigated by electrospray ionization tandem mass spectrometry (ESI‐MS/MS) in positive ion mode. In mass spectrometry of the enaminones, Ar? CO? CH?CH? N(CH₃)₂, the proton transfers from the thermodynamically favored site at the carbonyl oxygen to the dissociative protonation site at ipso‐position of the phenyl ring or the double bond carbon atom adjacent to the carbonyl leading to the loss of a benzene or elimination of C₄H₉N, respectively. And the hydrogen? deuterium (H/D) exchange between the added proton and the proton of the phenyl ring via a 1,4‐H shift followed by hydrogen ring‐walk was witnessed by the D‐labeling experiments. The elemental compositions of all the ions were confirmed by ultrahigh resolution Fourier transform ion cyclotron resonance tandem mass spectrometry (FTICR‐MS/MS). The enaminones studied here were para‐monosubstituted on the phenyl ring and the electron‐donating groups were in favor of losing the benzene, whereas the electron‐attracting groups strongly favored the competing proton transfer reaction leading to the loss of C₄H₉N to form a benzoyl cation, Ar‐CO⁺. The abundance ratios of the two competitive product ions were relatively well‐correlated with the σ_p⁺ substituent constants. The mechanisms of these reactions were further investigated by density functional theory (DFT) calculations. Copyright © 2010 John Wiley & Sons, Ltd.     

Re-examination of the anion derivatives of isoflavones by radical fragmentation in negative electrospray ionization tandem mass spectrometry: experimental and computational studies

This paper reports theoretical and experimental studies of gas‐phase fragmentation reactions of four naturally occurring isoflavones. The samples were analyzed in negative ion mode by direct infusion in ESI‐QqQ, ESI‐QqTOF and ESI‐Orbitrap systems. The MS/MS and MSⁿ spectra are in agreement with the fragmentation proposals and high‐resolution analyses have confirmed the formulae for each ion observed. As expected, compounds with methoxyl aromatic substitution have showed a radical elimination of ?CH₃ as the main fragmentation pathway. A second radical loss (?H) occurs as previously observed for compounds which exhibit a previous homolytic ?CH₃ cleavage (radical anion) and involves radical resonance to stabilize the anion formed. However, in this study we suggest another mechanism for the formation of the main ions, on the basis of the enthalpies for each species. Compounds without methoxy substituent dissociate at the highest energies and exhibit the deprotonated molecule as the most intense ion. Finally, energy‐resolved experiments were carried out to give more details about the gas‐phase dissociation reaction of the isoflavones and the results are in agreement with the theoretical approaches. Copyright © 2011 John Wiley & Sons, Ltd.     

Multi-anode detection in electrospray ionization time-of-flight mass spectrometry

An electrospray ionization ion source coupled to a time-of-flight mass analyzer incorporating a multi-anode time-to-digital converter is described. High-speed data acquisition (kHz mass spectral acquisition) rates are achieved. The four-anode detector produces a significant increase in detection/counting efficiency over that for a single-anode detector. In this work a 2.5 times increase in detection efficiency is demonstrated. The multi-anode detector is also used as a diagnostic tool to optimize transmission of the ion optics.     

Nanoscale separations combined with electrospray ionization mass spectrometry: Sulfonamide determination

Nanoscale capillary liquid chromatography (nCLC) and capillary zone electrophoresis (CZE) have been combined with quadrupole mass spectrometry via an electrospray ionization (ESI) interface. These methodologies have been applied to the separation and determination of a variety of sulfonamides. CZE/ESI/MS is the more rapid and sensitive technique, but nCLC/ESI/MS shows promise for the analysis of dilute samples. Ultimately, the two techniques provide complementary methods of analysis. The detection limits of these techniques in the full-scan mode are in the low picomole range. Dissociation of the sulfonamides can be induced by increasing the skimmer voltage. This provides a limited means of discriminating between compounds of identical molecular weight but, more important, provides fragments that could be used to confirm the presence of analyte within a sample.     

Letter: Influence of experimental conditions on electrospray ionization mass spectrometry of ferrocenylalkylazoles

Influence of experimental conditions on electrospray/ionization (ESI) mass spectra of ferrocene derivatives FcCHRAz (Fc = eta5-C5H5-Fe-eta5- C5H4; R = H, Az = benzimidazole; R = Ph, Az = 2-methylimidazole) has been investigated. The spectra of all the compounds revealed [M]+*, product of its fragmentation [FcCHR]+ as well as products of ion/molecular interactions (protonated molecule [MH]+, binuclear ion [(FcCHR)2 Az]+, dimeric ion [M2]+* and its protonated form [M2H]+). Relative abundances of these ions appreciably (more than one order) depend on experimental conditions: analyte concentration, temperature of heated capillary, spray voltage, flow rate of mobile phase and polarity of solvents. Established correlations allow the selection of optimum experimental conditions for registration of ESI mass spectra, as required by the application. If an unknown compound is to be identified, it is necessary to operate by using polar solvents, small concentration, low temperature of heated capillary, high spray voltage and flow rates. There are high-intensity binuclear and protonated dimeric ions in mass spectra under other conditions. It can give rise to wrong interpretation of the structure of investigated compound. At the same time, for study of ion-molecular processes by ESI-MS it is necessary to use concentrated samples in non-polar solvents. In this case the dependence of reaction products yields on temperature and flow rate of mobile phase must be investigated.     

Exploring rearrangements along the fragmentation pathways of diuron anion: A combined experimental and computational investigation

Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), a common herbicide from phenyl urea class, was investigated by studying the formation of several negative ions [M−H]⁻ in the gas phase and the fragmentation behaviour of the thermodynamically most probably formed isomeric anions upon linear ion acceleration/collision experiments. The collision induced dissociation experiments (CID) were carried out in a hexapole–quadrupole–hexapole hybrid system coupled to 12 T magnet with infinity ICR cell for high resolution measurements. Two distinctive main pathways were observed in the MS/MS spectrum. Sustained off-resonance irradiation (SORI) experiments inside the ICR cell reinforce the fragmentation channels obtained from linear ion acceleration experiments. The fragmentation pathways were also completely investigated by the use of B3LYP/6-311+G(2d,p)//B3LYP/6-31+G(d) level of theory. Elimination of dimethylamine takes place in a two-step process, by which two successive 1,3 proton shifts occur. The second 1,3 proton shift is concerted with the departure of dimethylamine. The driving force for the (CH₃)₂NH elimination is the formation of isocyanate group. The formed primary product ion can further decompose to release HCl through a new transition state. A stable new aromatic product ion is formed with 10π electrons. Loss of C₃H₅NO neutral from another anionic isomer of the precursor ion was also observed and is characteristic for the amide terminal of the diamide functional group. A concerted mechanism is proposed, by which N–C bond breakage and cyclization of the eliminated neutral fragment C₃H₅NO takes place simultaneously to form 1-methyl-aziridin-2-one.     

Charge competition and the linear dynamic range of detection in electrospray ionization mass spectrometry

An experimental investigation and theoretical analysis are reported on charge competition in electrospray ionization (ESI) and its effects on the linear dynamic range of ESI mass spectrometric (MS) measurements. The experiments confirmed the expected increase of MS sensitivities as the ESI flow rate decreases. However, different compounds show somewhat different mass spectral peak intensities even at the lowest flow rates, at the same concentration and electrospray operating conditions. MS response for each compound solution shows good linearity at lower concentrations and levels off at high concentration, consistent with analyte "saturation" in the ESI process. The extent of charge competition leading to saturation in the ESI process is consistent with the relative magnitude of excess charge in the electrospray compared to the total number of analyte molecules in the solution. This ESI capacity model allows one to predict the sample concentration limits for charge competition and the on-set of ionization suppression effects, as well as the linear dynamic range for ESI-MS. The implications for quantitative MS analysis and possibilities for effectively extending the dynamic range of ESI measurements are discussed.     

啤酒中单糖的衍生化HPLC-ESI-MS测定方法研究

单糖类样品在溶液中非常稳定,难于离子化,不适合于进行ESI-MS检测。采用1-苯基-3-甲基-5-吡唑啉酮(PMP)将糖类物质衍生化,HPLC-ESI-MS在线联用,选择性离子扫描方式对几种啤酒样品中的5种单糖进行了分离检测。检出限可达到80pg。     

Lariat ethers in the chiral recognition of amino acid esters:electrospray ionization mass spectrometry investigation

The ability of the crown ethers (1–4), containing the ortho- or para- methoxyphenoxy-methyl substituents in their structure, to chiral recognition in reference to amino acid esters has been investigated by electrospray ionization mass spectrometry (ESI-MS). The method allows registering the diastereomeric complexes between the studied crowns as hosts and the protonated alanine, phenylglycine and phenylalanine methyl esters as guests in the gas phase. ESI-MS experiments using isotopically labeled guests provide robust and reproducible results, indicating a moderate degree of chiral discrimination in the series of the studied crown ethers. ESI-MS experiments using achiral amine as a reference yielded the results comparable with the previous method. It has been found that (S)-enantiomers of the crowns bind predominately (S)-enantiomers of the amino acid esters, and vice-versa. It has been shown that the chiral ortho-substituted crown (S)-1 demonstrates the more pronounced values for chiral discrimination as compared with the para-substituted crown (S)-2. This fact indicates the interrelationship between the chiral recognition and the lariat nature of crown 1. Increasing the size of the cavity and the presence of a flat aromatic moiety in crowns 3 and 4 strengthens their complexing ability, simultaneously weakening the enantioselectivity of the complexation.     

Reactive extractive electrospray ionization tandem mass spectrometry for sensitive detection of tetrabromobisphenol A derivatives

Sensitive detection of tetrabromobisphenol A (TBBPA) and its derivatives, a group of emerging toxic contaminants, is highly necessitated in environmental investigation. Herein a novel analytical strategy based on reactive extractive electrospray ionization (EESI) tandem mass spectrometry for detection of tetrabromobisphenol A bis(2-hydroxyethyl ether) (TBBPA-BHEE), tetrabromobisphenol A bis(glycidyl ether) (TBBPA-BGE), tetrabromobisphenol A bis(allylether) (TBBPA-BAE), and tetrabromobisphenol S bis(allylether) (TBBPS-BAE) in industrial waste water samples was developed. Active silver cations (Ag⁺), generated by electrospraying a silver nitrate methanol solution (10 mg L⁻¹), collides the neutral TBBPA derivatives molecules in the EESI source to form [M + Ag]⁺ complexes of the analytes under the ambient conditions. Upon collision-induced dissociation (CID), characteristic fragments of the [M + Ag]⁺ complexes were identified for confident and sensitive detection of the four TBBPA derivatives. Under the optimized experimental conditions, the instrumental limits of detection (LODs) of TBBPA-BHEE, TBBPA-BGE, TBBPA-BAE and TBBPS-BAE were 0.37, 0.050, 0.76, and 4.6 μg L⁻¹, respectively. The linear ranges extended to 1000 μg L⁻¹ (R² ≥ 0.9919), and the relative standard deviations (RSDs), inter-day variation and intra-day variation were less than 7.8% (n = 9), 10.0% (n = 5), and 14.8% (n = 1 per day for 5 days) for all derivatives. TBBPA derivative manufacturing industrial waste water, river water and tap water samples were fast analyzed with the proposed method. The contents of TBBPA derivatives were various in the collected samples, with the highest 19.9 ± 0.3 μg L⁻¹ of TBBPA-BAE in the waste water samples.     

A computational and experimental study of cation affinity (Na+) of nucleobases and modified nucleobases by electrospray ionization ion trap mass spectrometry

Gas-phase Na+ affinities of modified or unmodified nucleobases were determined theoretically at the density functional theory level, with the B3P86 functional and the 6-31 + G* basis set, and experimentally using electrospray ionization ion trap mass spectrometry (ESI-ITMS) and the kinetic method. For the calculations, the sodium cation affinities (SCA) were obtained from energies of the most stable complexes of the free nucleobases. Experimentally and theoretically relative scales of cation affinities were determined using eight modified and unmodified nucleobases and a very good agreement was obtained.     

Direct detection of chloramphenicol in honey by neutral desorption-extractive electrospray ionization mass spectrometry

Herein, we constructed a platform of neutral desorption-extractive electrospray ionization mass spectrometry (ND-EESI-MS) for direct and rapid detection of chloramphenicol (CAP) in honey samples diluted with methanol. Under the optimized working conditions, the quantitative information of CAP residues was acquired effectively by EESI-Ion Trap MS ⁿ . Using heated methanol-N₂ as spray reagent, we reduced the limit of determination (LOD) from 73.3 ng/mL to 0.3 ng/mL, and the CAP detection is linear in the range of 1–5000 ng/mL (R?=?0.9947). For the honey samples with CAP of 10, 100, and 1000 ng/mL, the recoveries were 133.0, 80.6, and 101.1 %, and the relative standard deviations were 5.96, 8.82, and 8.71 %, respectively. The reproducibility assays showed the stability of this method. Therefore, this ND-EESI-MS method is powerful for direct, rapid, and quantitative CAP analysis in honey samples with high sensitivity, precision, and specificity. Graphical Abstract

In the current neutral desorption-extractive electrospray ionization mass spectrometry (ND-EESI-MS) method, N₂ is inlet into samples to desorb chloramphenicol (CAP). We tried to use some organic solvents as the spray reagent to dissolve CAP, and then the best neutral desorption efficiency was obtained when using methanol. We applied this modified ND-EESI-MS method to detect CAP in honey samples only with sample dilution. The limit of CAP detection was then reduced from 73.3 to 0.3 ng/mL, reaching the current EU standard. Therefore, this is a powerful method for direct, rapid, and quantitative CAP analysis in honey samples.     

Analytical application of acetate anion in negative electrospray ionization mass spectrometry for the analysis of triterpenoid saponins--ginsenosides

Ginsenosides containing different numbers of glycosyl groups can be easily differentiated based on the formation of characteristic ginsenoside-acetate adduct anions and deprotonated ginsenosides generated by electrospray ionization (ESI) of methanolic solutions of ginsenosides (M) and ammonium acetate (NH4OAc). Ginsenosides containing two glycosyl groups gave a characteristic mass spectral pattern consisting of [M+2OAc]2-, [M-H+OAc]2- and [M-2H]2- ions with m/z values differing by 30 Th, while this mass spectral pattern was not observed for ginsenosides containing one glycosyl group. Formation of [M+2OAc]2- was influenced by the chain length of glycosyl groups and was used to differentiate the ginsenosides containing different combinations of monosaccharide and disaccharide units in the glycosyl groups. Under identical collisional activation conditions, [M+OAc]-, [M-H+OAc]2- and [M+2OAc]2- underwent proton abstractions predominantly to generate [M-H]-, [M-2H]2- and [M-H+OAc]2- ions, respectively. The ion intensity ratios, I[M-H](-/I) [M+OAc]-, I[M-2H](2-/I) [M-H+2OAc]2- and I[M-H+OAc](2-/I) [M+OAc]2-, being sensitive to the structural differences of ginsenosides, could differentiate the isomeric ginsenosides, including (i) Rf, F11 and Rg1, (ii) Rd and Re, and (iii) Rb2 and Rc. Additionally, NH4OAc was found to enhance the sensitivity of detection of ginsenosides in the form of [M-H]- down to the femtomole level.     

A snapshot of enzyme catalysis using electrospray ionization mass spectrometry

Insights into the early molecular events involving protein-ligand/substrate interactions such as protein signaling and enzyme catalysis can be obtained by examining these processes on a very short, millisecond time scale. We have used time-resolved electrospray mass spectrometry to delineate the catalytic mechanism of a key enzyme in bacterial lipopolysaccharide biosynthesis, 3-deoxy-d-manno-2-octulosonate-8-phosphate synthase (KDO8PS). Direct real-time monitoring of the catalytic reaction under single enzyme turnover conditions reveals a novel hemiketal phosphate intermediate bound to the enzyme in a noncovalent complex that establishes the reaction pathway. This study illustrates the successful application of mass spectrometry to reveal transient biochemical processes and opens a new time domain that can provide detailed structural information of short-lived protein-ligand complexes.     

Use of electrospray ionization mass spectrometry for the investigation of radical cation chain reactions in solution: detection of transient radical cations

Electrospray ionization mass spectrometry (ESI-MS) coupled to a microreactor is an excellent tool for the investigation of reactions in solution. Here, we report the first results of our investigations into preparatively interesting electron-transfer-initiated chain reactions in solution which proceed via radical cations as reactive intermediates. The tris(p-bromophenyl)aminium hexachloroantimonate (1)-mediated [2+2] cycloaddition of trans-anethole (2) to give 1,2-bis(4-methoxyphenyl)-3,4-dimethylcyclobutane (3) was investigated. The reaction proceeds as a radical cation chain reaction via transient intermediates 2 ⁺ and 3 ⁺ that could be detected and characterized unambiguously directly in the reacting solution by ESI-MS/MS. The identity of the intermediates was confirmed by comparison with authentic MS/MS spectra of 2 ⁺ and 3 ⁺ obtained by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). In addition, substrate and product can be monitored easily in the reacting solution by APCI-MS.Part of this work was presented at the 36th Conference of the German Society for Mass Spectrometry (DGMS).     

High-throughput detection of drugs binding to proteins using desorption electrospray ionization mass spectrometry

In this paper, we present a strategy for screening drugs that bind to proteins by combining centrifugal filtration with desorption electrospray ionization mass spectrometry (DESI-MS). Membrane filtration was used to remove any unbound drugs. Then, drug–protein complexes deposited on the DESI substrate were dissociated during the DESI-MS analytical process, and the liberated drugs were measured. To validate the methodology, the screening of a series of drugs against two types of proteins was performed. Three DNA topoisomerase I (Topo I) inhibitors (camptothecin (CPT), hydroxycamptothecin (OHCPT) and 7-ethyl-10-hydroxycamptothecin (SN-38)) were screened against Topo I and the DNA-Topo I complex using DESI-MS. The results indicated that none of the inhibitors bound to Topo I, because the inhibitors had binding affinities only to the DNA-Topo I complex. Among the three drugs that bound to the DNA-Topo I complex, SN-38 had the strongest relative binding affinity, and CPT had the weakest relative binding affinity. The impact of the DESI spray solvent composition on the analysis of drug–protein complex binding was evaluated. Seven alkaloid drugs were also screened against Topo I using DESI-MS. Berberine and palmatine had good binding affinities. A screening of 21 types of drugs was carried out to determine whether the drugs bound to human serum albumin (HSA). The DESI-MS screening process could be achieved within 1.75 min. The study provides a method to qualitatively detect compounds that bind to proteins, showing great potential in drug design and screening.     

Direct detection of native proteins in biological matrices using extractive electrospray ionization mass spectrometry

The high-throughput and sensitive characterization of native proteins in biological samples is of increasing interest in multiple disciplines. Extractive electrospray ionization (EESI) forms ions of native proteins including lysozyme, α-chymotrypsin, myoglobin, human serum albumin, RNAse A and blood hemoglobin in extremely complex biosamples or PBS buffer solutions by softly depositing charges on the protein molecules. This method produces no significant conformational changes of the proteins in the ion formation process, and features direct detection of trace proteins present in biological matrices. The detection limit of low pmol L(-1) for lysozyme in untreated biological liquids such as human urine and tears was demonstrated using EESI mass spectrometry (MS), showing an attractive MS platform for the direct analysis of native proteins in actual biological samples.     

Fast quantitative detection of cocaine in beverages using nanoextractive electrospray ionization tandem mass spectrometry

Without any sample pretreatment, effervescent beverage fluids were manually sprayed into the primary ion plume created by using a nanoelectrospray ionization source for direct ionization, and the analyte ions of interest were guided into an ion trap mass spectrometer for tandem mass analysis. Functional ingredients (e.g., vitamins, taurine, and caffeine, etc.) and spiked impurity (e.g., cocaine) in various beverages, such as Red Bull energy drink, Coco-cola, and Pepsi samples were rapidly identified within 1.5 s. The limit of detection was found to be 7 ∼ 15 fg (S/N = 3) for cocaine in different samples using the characteristic fragment (m/z 150) observed in the MS³ experiments. Typical relative standard deviation and recovery of this method were 6.9% ∼ 8.6% and 104% ∼ 108% for direct analysis of three actual samples, showing that nanoextractive electrospray ionization tandem mass spectrometry is a useful technique for fast screening cocaine presence in beverages.     

Rapid analysis of nucleotide-activated sugars by high-performance liquid chromatography coupled with diode-array detection, electrospray ionization mass spectrometry and nuclear magnetic resonance

A generally applicable method for HPLC analysis of sugar nucleotides was established. Separation was achieved using ion-pair chromatography on a reversed-phase column. Ion-pair reagents were selected and various parameters optimized with respect to separation of 11 of the most important sugar nucleotides and compatibility with on-line detection by electrospray ionization MS and NMR. The method was applied to the on-line analysis of the GDP-D-mannose-4,6-dehydratase (Gmd) and GDP-4-keto-6-deoxy-D-mannose reductase (Rmd) catalyzed conversion of GDP-D-mannose to GDP-D-rhamnose. By LC-NMR, the intermediate product of the reaction was shown to be a mixture of GDP-4-keto-6-deoxy-D-mannose and GDP-3-keto-6-deoxy-D-mannose. Nucleotide co-factors of enzymatic reactions such as ATP and NADH did not interfere with the analysis of nucleotide-activated sugars.