Synthesis and mass spectral fragmentations of new spiro heterocycles

The reaction of dichlorodiphenoxymethane (2) with ortho-functionlized benzoic acids 1a-c yielded the spiro derivatives of bezodioxinone 4a, benzoxazinone 4b and benzodioxepinone 4c. The same reaction with 1,8-diaminonaphthalene afforded the spiro perimidine 6, while with 1,1'-binaphthy1-2,2'-diol gave the semi-cyclized derivative 2,2-diphenoxydinaphthodioxepine 8.     

Effect of mass transfer and solution composition on the quantification of reaction kinetics by differential electrochemical mass spectrometry

Differential electrochemical mass spectrometry(DEMS)is one of the most powerful techniques for both the mechanistic and kinetic study of complicated electrocatalytic reactions.It can provide information on the nature and yields of the products generated,their production rate,and the structure-activity relationship between the electrocatalysts and the target reactions.The precise calibration of the mass signal is a prerequisite for the accurate evaluation of reaction kinetics.In this work,we use the oxidation reactions of CO and HCOOH to demonstrate how certain conditions,such as the flow rate and solution composition,affect the collection efficiency and ionization probability of the species to be detected.These parameters can affect the determination of the mass calibration constant and the accuracy of the subsequent quantitative DEMS analysis.We show the relationship between the mass calibration constant and the flow rate,and provide strategies for eliminating this and the related problems.     

[M + Fe − 5H]2− peptide ion composition verified by Fourier transform mass spectrometry accurate mass and tandem mass spectrometry analyses

Previously, the unusual ion composition [M + Fe - 5H]2- had been proposed as the major species observed when a gamma-carboxy glutamate-containing glyco-peptide was analyzed with electrospray ionization in the negative ionization mode. The sequence assignment of this highly post-translationally modified peptide was based on the mass analysis using a quadrupole ion trap together with information from both Edman and DNA sequencing. Because there was little precedent for the loss of five protons from a ferric cationized peptide, we utilized Fourier transform mass spectrometry accurate mass and tandem mass spectrometry analyses to verify the peptide ion composition.     

Polyatomic ions in thermal ionisation mass spectrometry — challenges and opportunities

Polyatomic ions, often considered as causing interference in ICP-MS, SSMS and GDMS, are useful in thermal ionisation mass spectrometry (TIMS) for determining the atomic ratios of the elements, particularly for light elements. The objective of this paper is to provide a detailed discussion on the handling of the isotopic measurement data in TIMS using polyatomic ions, a useful technique for light elements, to reduce isotope fractionation effects. Taking as an example the Li₂BO₂⁺ ion for the determination of the ⁶Li/⁷Li or ¹⁰B/¹¹B ratio of the unknown sample, a detailed theoretical analysis is presented for optimum selection of the pair of polyatomic ions to be used to determine the isotopic ratio of the element. The theory is supported by experimental data from the literature in three different examples: (i) the isotopic analysis of natural Li samples using the SRM-951-B isotopic standard; (ii) the isotopic analysis of an enriched ⁶Li sample using SRM-951-B; (iii) the isotopic analysis of an enriched ¹⁰B sample using natural Li (Svec standard). It is shown that the four polyatomic peaks observed in the m/z range of 54–57 are of practical importance. A qualitative idea can be obtained about the isotopic composition of Li and B in the sample (natural or enriched) based on the intensity distribution of these four peaks in the mass spectrum. When calculating accurate atomic ratios from the observed intensities of the polyatomic peaks, a simple “rule of thumb” should be kept in mind: the polyatomic ratio that is closer to the expected atomic ratio provides an accurate value of the atomic ratio of the element in the unknown sample. Even between the two polyatomic ion ratios, better accuracy is possible in cases which do not magnify the error during calculation and show less isotopic fractionation in the ion source. It has been stressed that the two peaks of highest intensity in the polyatomic ion are not necessarily the best for arriving at atomic ratios during the analyses of unknown samples, for depleted as well as enriched ⁶Li and ¹⁰B samples.     

High-resolution Orbitrap mass spectrometry for the analysis of carotenoids in tomato fruit: validation and comparative evaluation towards UV–VIS and tandem mass spectrometry

In this study, a generic extraction protocol and full-scan high-resolution Orbitrap-mass spectrometry (MS) detection method were developed, enabling the metabolomic screening for carotenoids in tomato fruit tissue. To this end, the carotenoids lutein, zeaxanthin, α-carotene, β-carotene, and lycopene (representing both xanthofylls and carotenes) were considered. The extraction procedure was optimized by means of a D-optimal design and consisted of a liquid–liquid extraction with methanol/tert-butyl methyl ether (1:1, v/v). The considered compounds were detected by a single-stage Exactive^TM mass spectrometer, operating at a mass resolution of 100,000 full width at half maximum. The validation study demonstrated excellent performance in terms of linearity (R ²?>?0.99), repeatability (CV?≤?10.6 %), within-laboratory reproducibility (CV?≤?12.2 %), and mean corrected recovery (ranging from 85 to 106 %). Additionally, a comparative evaluation towards well-established detection techniques, i.e., tandem mass spectrometry (MS/MS) and ultraviolet-visible spectroscopy (UV–VIS) photodiode array, indicated superior performance of high-resolution Orbitrap-MS with regard to specificity/selectivity and sensitivity (with limits of detection ranging from 1.0 to 3.8 pg μL^?1). As a result, it may be concluded that high-resolution Orbitrap-MS is a suited alternative for UV–VIS or MS/MS in analyzing carotenoids and may offer significant value in carotenoid research because of the metabolomic screening possibilities.

Optimization of antibiotic analysis in water by solid-phase extraction and high performance liquid chromatography–mass spectrometry/mass spectrometry

This paper describes the development of an optimized method based on solid-phase extraction (SPE) followed by liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS) for the simultaneous analysis of ten antibiotic compounds including tetracyclines, sulfonamides, macrolides and quinolones. LC–MS/MS sensitivity has been optimized by alterations to both LC and MS operations. Of the two high resolution columns tested, Waters Symmetry C₁₈ endcapped and Agilent Zorbax Bonus-RP, the latter was found to show better performance in producing sharp peaks and clear separation for most of the target compounds. Optimization of the MS fragmentation collision and cone energy enhanced the peak areas of the target analytes. The recovery of the target compounds from water samples was most efficient on Waters Oasis HLB SPE cartridge, while methanol was shown to be the most suitable solvent for desorbing the compounds from SPE. In addition, acidification of samples prior to SPE was shown to enhance the recovery of the compounds. To ensure a satisfactory recovery, the flow rate through SPE should be maintained at ≤10 mL min⁻¹. The method was successfully applied to the analysis of antibiotics from environmental water samples, with concentrations being <LOD in tap water, between <LOD to 28 ng L⁻¹ in river water and between <LOD to 230 ng L⁻¹ in sewage effluent.     

Comparison of supercritical fluid chromatography–tandem mass spectrometry and liquid chromatography–tandem mass spectrometry for the stereoselective analysis of chlorfenvinphos and dimethylvinphos in tobacco

This study used reversed-phase liquid chromatography–tandem mass spectrometry and supercritical fluid chromatography–tandem mass spectrometry for determination of the stereoisomers of chlorfenvinphos and dimethylvinphos in tobacco. Tobacco samples were extracted and purified with a modified quick, easy, cheap, effective, rugged, and safe technique using spherical carbon. The performance of both methodologies was comprehensively compared in terms of methods validation parameters (separation efficiency, linearity, selectivity, recovery, repeatability, sensitivity, matrix effect, etc.). Under optimized conditions, the calibration curves of the stereoisomers of chlorfenvinphos and dimethylvinphos in the range of 10–500 ng/mL showed excellent linearity with R² ≥ 0.997 in both methods. The adequate recoveries of analytes from three different spiked tobaccos were obtained using reversed-phase liquid chromatography–tandem mass spectrometry (86.1–95.7%) as well as supercritical fluid chromatography–tandem mass spectrometry (86.5–94.0%). The relative standard deviations for spiked samples were all below 7.0%. Compared with supercritical fluid chromatography–tandem mass spectrometry, lower matrix effects and LODs can be obtained in reversed-phase liquid chromatography–tandem mass spectrometry.     

Ionization and transmission efficiency in an electrospray ionization—mass spectrometry interface

The ionization and transmission efficiencies of an electrospray ionization (ESI) interface were investigated to advance the understanding of how these factors affect mass spectrometry (MS) sensitivity. In addition, the effects of the ES emitter distance to the inlet, solution flow rate, and inlet temperature were characterized. Quantitative measurements of ES current loss throughout the ESI interface were accomplished by electrically isolating the front surface of the interface from the inner wall of the heated inlet capillary, enabling losses on the two surfaces to be distinguished. In addition, the ES current lost to the front surface of the ESI interface was spatially profiled with a linear array of 340-microm-diameter electrodes placed adjacent to the inlet capillary entrance. Current transmitted as gas-phase ions was differentiated from charged droplets and solvent clusters by measuring sensitivity with a single quadrupole mass spectrometer. The study revealed a large sampling efficiency into the inlet capillary (>90% at an emitter distance of 1 mm), a global rather than a local gas dynamic effect on the shape of the ES plume resulting from the gas flow conductance limit of the inlet capillary, a large (>80%) loss of analyte ions after transmission through the inlet arising from incomplete desolvation at a solution flow rate of 1.0 microL/min, and a decrease in analyte ions peak intensity at lower temperatures, despite a large increase in ES current transmission efficiency.     

Relevance and use of capillary coatings in capillary electrophoresis–mass spectrometry

Over the last two decades, coupled capillary electrophoresis (CE)–mass spectrometry (MS) has developed into a generally accepted technique with a wide applicability. A growing number of CE-MS applications make use of capillaries where the internal wall is modified with surface coating agents. In CE-MS, capillary coatings are used to prevent analyte adsorption and to provide appropriate conditions for CE-MS interfacing. This paper gives an overview of the various capillary coating strategies used in CE-MS. The main attention is devoted to the way coatings can contribute to a proper CE-MS operation. The foremost capillary coating methods are discussed with emphasis on their compatibility with MS detection. The role of capillary coatings in the control of the electroosmotic flow and the consequences for CE-MS coupling are treated. Subsequently, an overview of reported applications of CE-MS employing different coating principles is presented. Selected examples are given to illustrate the usefulness of the coatings and the overall applicability of the CE-MS systems. It is concluded that capillary coatings can enhance the performance and stability of CE-MS systems, yielding a highly valuable and reproducible analytical tool.     

Evaluating reproducibility and similarity of mass and intensity data in complex spectra—applications to tubulin

We present a data processing approach based on the spectral dot product for evaluating spectral similarity and reproducibility. The method introduces 95% confidence intervals on the spectral dot product to evaluate the strength of spectral correlation; it is the only calculation described to date that accounts for both the non-normal sampling distribution of the dot product and the number of peaks the spectra have in common. These measures of spectral similarity allow for the recursive generation of a consensus spectrum, which incorporates the most consistent features from statistically similar replicate spectra. Taking the spectral dot product and 95% confidence intervals between consensus spectra from different samples yields the similarity between these samples. Applying the data analysis scheme to replicates of brain tubulin CNBr peptides enables a robust comparison of tubulin isotype expression and post-translational modification patterns in rat and cow brains.     

Amino acid identification and sequence analysis of peptides by reaction mass spectrometry

Fast atom bombardment mass spectrometry (FAB-MS) is applied to distinguish N-terminal series ions from C-terminal series ions of a peptide by on-probe acetylation, it providesvaluable information about the sequence of an unknown peptide. The FAB mass spectra containa number of characteristic ions at low-mass region in addition to the sequence ions at high-massregion. It was found that the ions below m/z 200 are characteristic of the amino acid composition ofthe peptide, from which the amino acid composition of the peptide could be estimated. Additionally,mixture analysis is also discussed.     

Development of a liquid–chromatography tandem mass spectrometry and ultra-high-performance liquid chromatography high-resolution mass spectrometry method for the quantitative determination of zearalenone and its major metabolites in chicken and pig plasma

A sensitive and specific method for the quantitative determination of zearalenone (ZEN) and its major metabolites (α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), α-zearalanol (α-ZAL), β-zearalanol (β-ZAL) and zearalanone (ZAN)) in animal plasma using liquid chromatography combined with heated electrospray ionization (h-ESI) tandem mass spectrometry (LC–MS/MS) and high-resolution Orbitrap^® mass spectrometry ((U)HPLC–HR–MS) is presented. The sample preparation was straightforward, and consisted of a deproteinization step using acetonitrile. Chromatography was performed on a Hypersil Gold column (50 mm × 2.1 mm i.d., dp: 1.9 μm, run-time: 10 min) using 0.01% acetic acid in water (A) and acetonitrile (B) as mobile phases.     

Mobile phase selection for the combined use of liquid chromatography–inductively coupled plasma mass spectrometry and electrospray ionisation mass spectrometry

Four different organic solvents: dimethylformamide, 1,4-dioxane, n-propanol and ethanol were evaluated as alternative organic modifiers to acetonitrile for liquid chromatography (LC) separations. The aim was to establish common sets of chromatographic conditions that could be applied for LC hyphenation to inductively coupled plasma mass spectrometry (ICPMS) as well as to electrospray ionization MS (ESIMS). The approach was to evaluate candidate solvents that, compared to acetonitrile, potentially could give improved analytical performance (low solvent vapor loading, maximized analyte sensitivity and minimized carbon depositions on instrumental parts) in ICPMS analysis while retaining chromatographic and ESIMS performances. The study showed that dimethylformamide, 1,4-dioxane, n-propanol and ethanol all can be advantageous chromatographic modifiers for LC–ICPMS analysis, giving superior performance compared to acetonitrile. For the combined use of LC–ICPMS and LC–ESIMS with a common set of chromatographic conditions, n-propanol gave the best overall performance. The ¹⁹⁵Pt⁺ signal in ICPMS was continuously monitored during a 0–60% organic solvent gradient and at 25% of organic modifier, 100% of the signal obtained at the gradient start was preserved for n-propanol compared to only 35% of the signal when using acetonitrile. Platinum detection limits were 5–8 times lower using n-propanol compared with acetonitrile. Signal-to-noise ratio in continuous ESIMS signal measurements was 100, 90 and 110 for a 100 μg/ml solution of leucine–enkephaline using acetonitrile, ethanol and n-propanol, respectively. Chromatographic efficiency in reversed phase separations was preserved for n-propanol compared to acetonitrile for the analysis of the whole protein cytochrome C and the peptide bacitracin on a column with particle and pore sizes of 5 μm and 300 Å, but slightly deteriorated for the separation of the peptides leucine–enkephaline and bacitracin on a 3 μm and 90 Å column as the peak width at half height for both peptides increased by a factor of two. The performance on the smaller dimensioned column could however be improved by running the separations at 40 °C.     

Identification and determination of mycotoxins and food additives in feed by HPLC–high-resolution time-of-flight mass spectrometry

High-resolution time-of-flight mass spectrometry combined with high performance liquid chromatography is proposed for the detection and determination of 25 mycotoxins and 8 food additives (coccidiostats) in animal feed, using simplified and rapid sample preparation. We developed a procedure for the identification and determination of analytes by the standard addition method. The lower limit of the analytical range is 1 (400) µg/kg for mycotoxins; the analytical range for coccidiostats in feed is 10–200 mg/kg. The relative standard deviation of the results does not exceed 10%. The analysis time is 0.5–1 h.     

Nontargeted screening of veterinary drugs and their metabolites in milk based on mass defect filtering using liquid chromatography–high-resolution mass spectrometry

The development of nontargeted screening strategy for veterinary drugs and their metabolites is very important for food safety. In this study, a nontargeted screening strategy was developed to find the potentially hazardous substances based on mass defect filtering (MDF) using liquid chromatography–high-resolution mass spectrometry. First, the drug metabolites of 112 veterinary drugs from seven classes of antimicrobials were predicted. Second, three MDF models were established, including the traditional rectangular MDF, the enhanced parallelogram MDF, and the polygonal MDF. Finally, the strategy was applied to nontargeted screening of veterinary drugs in 36 milk samples. The polygonal MDF model based on the distribution area of parent drugs and their metabolites showed a better filtering effect. After removing food components and performing MDF, about 10% of the substances remained, and four veterinary drugs and six drug metabolites were discovered and identified, showing the effectiveness of this strategy. The nontargeted screening strategy can rapidly remove interfering substances and find the suspected compounds. It can also be used for nontargeted screening of veterinary drugs and their metabolites in other food matrices.     

Sulfated and sulfonated polysaccharide as chiral stationary phases for capillary electrochromatography and capillary electrochromatography–mass spectrometry

The applications of polysaccharide phenyl carbamate derivatives as chiral stationary phases (CSPs) for capillary electrochromatography (CEC) are often hindered by longer retention times, especially using a normal-phase (NP) eluent due to very low electroosmotic flow (EOF). Therefore, in this study, we propose an approach for the aforementioned problems by introducing two new types of negatively charged sulfate and sulfonated groups for polysaccharide CSPs. These CSPs were utilized to pack CEC columns for enantioseparation with a NP eluent. Compared to conventional cellulose tris(3,5-dimethylphenyl carbamate) or CDMPC CSPs, the sulfated CDMPC CSP (sulfur content 4.25%, w/w) shortened the analysis time up to 50% but with a significant loss of enantiomeric resolution (∼60%). On the other hand, the sulfonated CDMPC CSP (sulfur content 1.76%, w/w) not only provided fast throughput but also maintained excellent resolving power. In addition, its synthesis is much more straightforward than the sulfated one. Furthermore, we studied several stationary phase parameters (CSP loading and silica gel pore size) and mobile phase parameters (including type of mobile phase and its composition) to evaluate the throughput and enantioselectivity. Using the optimized conditions, a chiral pool containing 66 analytes was screened to evaluate the enantioselectivity under three different mobile phase modes (i.e., NP, polar organic phase (POP) and reversed-phase (RP) eluents). Among these mobile phase modes, the RP mode showed the highest success rate, whereas some degree of complementary enantioselectivity was observed with NP and POP. Finally, the feasibility of applying this CSP for CEC–MS enantioseparation using internal tapered column was evaluated with NP, POP and RP eluents. In particular, the NP-CEC–MS provided significantly enhanced sensitivity when methanol was replaced with isopropanol in the sheath liquid. Using aminoglutethimide as model chiral analyte, all three modes of CEC–MS demonstrated excellent durability as well as excellent reproducibility of retention time and enantioselectivity.     

Analysis of dinitro- and amino-nitro-toluenesulfonic acids in groundwater by solid-phase extraction and liquid chromatography–mass spectrometry

A reversed-phase LC–MS method with quadrupole-time of flight (QTOF) detection has been developed for the determination of four dinitro-toluenesulfonic acids and two amino-nitro-toluenesulfonic acids in groundwater. The analytes were separated by HPLC with 0.1% (v/v) formic acid as mobile phase modifier compatible with mass spectrometric detection. QTOF-MS analysis with negative ion electrospray ionization afforded good selectivity and sensitivity for analysis of the dinitro- and amino-nitro-toluenesulfonic acids. Structure elucidation and confirmation were accomplished by tandem mass spectrometry. Characteristic ions resulting from the loss of NO, NO₂, and SO₂ from the [M–H]^– ions were detected. An intense fragment ion at m/z 80 representing the [SO₃]^– ion was detected for all dinitro- and amino-nitro-toluenesulfonic acids. Solid-phase extraction using a co-polymer cartridge was developed for preconcentration of the analytes from water. Good recovery (>85%) was achieved when 0.1% formic acid was added into the water samples before extraction. Method detection limits ranged from 10 to 76 ng L^–1 for the targeted compounds when 10 mL water was analyzed. Groundwater samples collected from wells close to a former ammunition plant in Stadtallendorf, Germany, were analyzed for the dinitro- and amino-nitro-toluenesulfonic acids.     

Detection and quantification of plasma amyloid-β by selected reaction monitoring mass spectrometry

Amyloid-β (Aβ) in human plasma was detected and quantified by an antibody-free method, selected reaction monitoring mass spectrometry (SRM-MS) in the current study. Due to its low abundance, SRM-based quantification in 10 μL plasma was a challenge. Prior to SRM analysis, human plasma proteins as a whole were digested by trypsin and high pH reversed-phase liquid chromatography (RPLC) was used to fractionate the tryptic digests and to collect peptides, Aβ_1–5, Aβ_6–16, Aβ_17–28 and Aβ_29–40(42) of either Aβ_1–40 or Aβ_1–42. Among those peptides, Aβ_17–28 was selected as a surrogate to measure the total Aβ level. Human plasma samples obtained from triplicate sample preparations were analyzed, obtaining 4.20 ng mL⁻¹ with a CV of 25.3%. Triplicate measurements for each sample preparation showed CV of <5%. Limit of quantification was obtained as 132 pM, which corresponded to 570 pg mL⁻¹ of Aβ_1–40. Until now, most quantitative measurements of Aβ in plasma or cerebrospinal fluid have required antibody-based immunoassays. Since quantification of Aβ by immunoassays is highly dependent on the extent of epitope exposure due to aggregation or plasma protein binding, it is difficult to accurately measure the actual concentration of Aβ in plasma. Our diagnostic method based on SRM using a surrogate peptide of Aβ is promising in that actual amounts of total Aβ can be measured regardless of the conformational status of the biomarker.     

Fast gel permeation chromatography–mass spectrometry method for polymer and additive analysis

This study presents gel permeation chromatography (GPC) coupled with mass spectrometry (MS) as a suitable method to evaluate molecular weight distribution, oligomeric structure, and additives of commercial polystyrene resins in just 4?min. The chromatogram recorded by ultraviolet (UV) detection gives information on the high molecular mass fractions, while the mass detector provides knowledge on the chemical structure and concentration of oligomers and additives. A good agreement for the average molecular weights of the broad polystyrene reference SRM 706 and an excellent correlation with the expected isotope distributions for oligomers and additives were obtained using this fast GPC–UV–MS method.