Portable electrospray ionization mass spectrometry (ESI-MS) for analysis of contaminants in the field

Hitherto analysis of chemicals in the field using mass spectrometry (MS) has been limited to analysis of non-polar and thermally stabile organic compounds using either a direct gas leak or a membrane inlet as MS interface. Recently, Professor R. Graham Cooks’ group demonstrated that miniature mass spectrometers operating at elevated pressures (>0.13?Pa (1?·?10^?3??Torr)) can be combined with electrospray ionization (ESI) for analysis of polar as well as thermally labile organic compounds. We present a simple miniaturized ESI unit for analysis of small liquid samples using miniature mass spectrometry. The ESI unit operates without pumps and supplementary sheath gases, which makes it very simple to handle in the field. 20–30?µL of sample solution is simply dropped into a small cavity in the ESI unit, where after the spray is initiated by applying high voltage to it. The miniaturized ESI unit was tested in combination with a miniature mass spectrometer (the Mini 10 developed by Professor R. Graham Cooks, Purdue University, IN) and we found that common herbicides (Atrazine, Prometryne, Terbutryne and Triadimefone) could be detected with detection limits around 1?mg?L^?1 and with a quantitative reproducibility of +/?30%. These characteristics, although high for environmental samples, are comparable to detection limits obtained with other ESI units used with miniature mass spectrometers and represent an early step forward towards a future field instrument. A major advantage of the capillary spray cell is its direct compatibility with micro extraction techniques for sample pre-concentration.     

Determination of binding sites in carboplatin-bound cytochrome c using electrospray ionization mass spectrometry and tandem mass spectrometry

Interaction of carboplatin with cytochrome c (Cyt. c) has been investigated by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS). ESI-MS studies revealed that the ring-opened adducts of carboplatin with Cyt. c were formed in the stoichiometric ratio of 1:1 and 2:1 at pH 5.0 and 37 degrees C and in the stoichiometric ratio of 1:1 only at pH 7.0 and 37 degrees C. It was also found that Cyt. c could be cleaved by carboplatin at pH 2.5 and 50 degrees C. The cleaved fragments of Cyt. c were determined by ESI-MS and MS/MS analysis to be Glu66 approximately Met80, Ac-Gly01 approximately Met65, Glu66 approximately Glu104, Ac-Gly01 approximately Met80 and Ile81 approximately Glu104. The carboplatin prefers to anchor to Met65 first, then to Met80. To further confirm the binding site of Met, AcMet-Gly was used as the model molecule to investigate its interaction with carboplatin and its hydrolysis reaction. On the basis of species detected during the reaction monitored by ESI-MS, a possible pathway of the cleavage reaction was proposed.     

Determination of cyclic structure for polydithiane using electrospray ionization mass spectrometry

Disulfide polymers obtained by ring‐opening polymerization have been considered to have a possibility of a cyclic catenane structure as judged from their test properties on the loss modulus and stress–strain. Electrospray ionization mass spectrometry (ESI‐MS) was used to detemine molecular structure of polydithiane and polyoxyethylene to find whether they are present as a cyclic or as a linear structure. The results indicated that the polydithiane possesses the cyclic structure, and analysis of the isotope distribution of the spectral ions further showed that the polymer consists entirely of the cyclic structure without contamination of a linear polymer. A linear chain polymer with a benzylmercaptan end group was synthesized, and the ESI‐MS analysis revealed that the polymer was a mixture of both the cyclic and the linear polymer. The cyclic polymer is probably formed by back‐biting of the highly reactive sulfur radicals that had been formed during the polymerization reaction. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 4403–4406, 2000     

Structural characterization of acetylpyridinium-ethyl pyruvate adducts by electrospray ionization mass spectrometry

The reactions of ethyl pyruvate with acetic anhydride and pyridine were studied by electrospray ionization mass spectrometry (ESI-MS). Ethyl 2-acetoxy-2-pyridiniumpropionate (1) (m/z 238) resulting from the reaction of the acetylpyridinium cation with ethyl pyruvate, and the adduct of ethyl 2-acetoxyacrylate with a pyridinium cation (2), bound together by non-covalent interactions (m/z 238), were identified by ESI-MS for the first time. Structures 1 and 2 cannot be distinguished, probably because one may be converted into the other and vice versa.     

Detection of histamine in beer by nano extractive electrospray ionization mass spectrometry

In this study, rapid quantitative detection of histamine in beer was achieved by using nano extractive electrospray ionization mass spectrometry (nano EESI‐MS) coupling with standard addition method. Based on the MS² experiment, histamine concentrations in three beer samples were determined to be 1.10 ± 0.12 µg/ml, 0.81 ± 0.09 µg/ml and 0.79 ± 0.09 µg/ml. The limit of detection for this method was calculated to be 0.02 µg/ml. These results show that this novel method can be used for direct, rapid and sensitive detection of histamine in beer without any tedious sample pretreatment. Copyright © 2014 John Wiley & Sons, Ltd.     

Ambient imaging mass spectrometry by electrospray ionization using solid needle as sampling probe

Although being an atmospheric pressure ion source, electrospray ionization (ESI) has rarely been used directly for ambient imaging mass spectrometry because the sample has to be introduced as liquid solution through the capillary. Instead of capillary, probe electrospray ionization (PESI), which has been developed recently, uses a solid needle as the sampling probe, as well as the electrospray emitter, and has been applied not only for liquid solutions but also for the direct sampling on wet samples. Biological tissues are composed of cells that contain 70–90% water, and when the surface is probed by the needle tip, the biological fluid adhering to the needle can be electrosprayed directly or assisted by additional solvent added onto the needle surface. Here, we demonstrate ambient imaging mass spectrometry of mouse brain section using PESI, incorporated with an auxiliary heated capillary sprayer. The solvent vapor generated from the sprayer condensed on the needle tip, re‐dissolving the adhered sample, and at the same time, providing an indirect means for needle cleaning. The histological sections were prepared by fixation using paraformaldehyde, and the spatial analysis was automated by maintaining an equal sampling depth into the sample in addition to raster scan. Phospholipids and galactosylceramides were readily detected from the mouse brain section in the positive ion mode, and were mapped with 60 µm lateral resolution to form mass spectrometric images. Copyright © 2009 John Wiley & Sons, Ltd.     

Monitoring of unfolding of metallo-proteins by electrospray ionization mass spectrometry

An electrospray ionisation (ESI) mass spectrometric method for the determination of the equilibrium constant and free energy (DeltaG) of protein unfolding was used to monitor the denaturation process at different pH of three metallo-proteins, i.e. wild-type copper azurin, zinc azurin and wild-type amicyanin. The time course of the unfolding process was followed by dissolving the proteins under denaturing conditions (methanol-water (1 : 1, v/v)) at different pH (2.5, 3.0, 3.5) and recording ESI spectra at time intervals. The spectra showed two series of peaks, corresponding to the native holo-protein and the unfolded apo-protein. From the intensity ratio of these two series of peaks at increasing time and at equilibrium, the equilibrium constants for the unfolding process for the three proteins could be determined. From these equilibrium constants a DeltaG degrees derivation was attempted. The DeltaG degrees values obtained decrease with decrease in pH, in agreement with the expected reduction of conformational stability of proteins at lower pH. The results obtained confirm that ESI-MS can be used for monitoring of unfolding process and to derive quantitative thermodynamic data.     

Relationships between structure,ionization profile and sensitivity of exogenous anabolic steroids under electrospray ionization and analysis in human urine using liquid chromatography–tandem mass spectrometry

The relationships between the ionization profile, sensitivity, and structures of 64 exogenous anabolic steroids (groups I–IV) was investigated under electrospray ionization (ESI) conditions. The target analytes were ionized as [M + H]⁺ or [M + H–nH₂O]⁺ in the positive mode, and these ions were used as precursor ions for selected reaction monitoring analysis. The collision energy and Q3 ions were optimized based on the sensitivity and selectivity. The limits of detection (LODs) were 0.05–20 ng/mL for the 64 steroids. The LODs for 38 compounds, 14 compounds and 12 compounds were in the range of 0.05–1, 2–5 and 10–20 ng/mL, respectively. Steroids including the conjugated keto‐functional group at C3 showed good proton affinity and stability, and generated the [M + H]⁺ ion as the most abundant precursor ion. In addition, the LODs of steroids using the [M + H]⁺ ion as the precursor ion were mostly distributed at low concentrations. In contrast, steroids containing conjugated/unconjugated hydroxyl functional groups at C3 generated [M + H ? H₂O]⁺ or [M + H ? 2H₂O]⁺ ions, and these steroids showed relatively high LODs owing to poor stability and multiple ion formation. An LC‐MS/MS method based on the present ionization profile was developed and validated for the determination of 78 steroids (groups I–V) in human urine. Copyright © 2015 John Wiley & Sons, Ltd.     

Gas chromatography electrospray ionization mass spectrometry analysis of trimethylsilyl derivatives

A method based on the analysis of trimethylsilyl (TMS) derivatives by capillary gas chromatography electrospray ionization mass spectrometry (GC–ESI/MS) was proposed. To improve separation, analytes were derivatized to their TMS derivative. During ESI analysis, TMS derivatives may hydrolyze back to their polar native form and are thus suitable for ESI analysis. Several types of analytes were studied to investigate the potential of the approach. Not all TMS derivatives hydrolyzed back to their native form as anticipated. Incomplete hydrolysis was observed for TMS‐organic acids and TMS‐nonchlorinated phenols. For TMS‐chlorophenols, the observation of only the [M ? H]^? ion suggested that these phenols were hydrolyzed back to their native form. For TMS‐beta agonists, the hydrolysis rate was low; therefore, the hydrolysis product was not detected. Both TMS‐chlorophenols and TMS‐beta agonists provide a sensitivity in the range of low parts per billion (0.25–5 ng/ml and 0.5–10 ng/ml respectively). Copyright © 2016 John Wiley & Sons, Ltd.     

Determination of pharmaceuticals in aqueous samples using positive and negative voltage switching microbore liquid chromatography/electrospray ionization tandem mass spectrometry

Analytical methods were developed for atorvastatin, novobiocin and roxithromycin using microbore liquid chromatography/electrospray ionization tandem mass spectrometry (microbore LC/ESI-MS/MS) in positive and negative voltage switching mode. Atorvastatin and roxithromycin require the positive-ion mode, whereas the negative-ion mode is required for the determination of novobiocin. Using the positive and negative voltage switching function, the three analytes were determined with one injection, and the time required was half that required using separately run positive- and negative-ion modes, without any reduction in sensitivity. A microbore LC column (100 x 1.0 mm i.d.) was chosen for chromatographic separation with mobile phase solvents acetonitrile and 10 mM aqueous ammonium acetate. The flow-rate was 0.1 ml min(-1) and the injection volume was 1 micro l. The analytes were quantified in the multiple reaction monitoring mode with external standards. By switching the positive and negative voltage, the three analytes were determined with a 4 min chromatographic run and with instrumental detection limits of 1-3 pg. This analytical method, using a microbore LC column combined with solid-phase extraction, was applied successfully to the determination of trace levels of the above pharmaceuticals in aqueous samples. Atorvastatin was detected in a sewage treatment plant final effluent.     

Characterization of synthetic nucleic acids by electrospray ionization quadrupole time-of-flight mass spectrometry

The potential of electrospray ionization quadrupole-quadrupole-time-of-flight mass spectrometry (ESI-QqTOF-MS) for the characterization of synthetic nucleic acids was evaluated. Oligonucleotides ranging in size from 12 up to 51 nucleotides were analyzed via direct infusion MS as well as via liquid chromatography (LC) online hyphenated to MS. These experiments proved the outstanding mass spectrometric performance of the TOF mass analyzer in regard of accuracy, reproducibility, resolution, and sensitivity. During a 1-min run, the monoisotopic mass of (dT)(24) was measured with a maximum relative mass deviation of 7.64 ppm proving the high mass accuracy of the TOF analyzer. Over a period of 1 h, mean deviations were determined in the range between -3.58 ppm and 3.06 ppm demonstrating the high stability of the applied external calibration. The molecular mass of a 51-mer was measured with a deviation smaller than 3.23 ppm from the theoretical value. The resolution exceeded a value of m/Deltam = 20 000 (m is the measured mass and Deltam the full peak width at half-maximum), which enabled the separation of the isotopic peaks of all investigated oligonucleotides. Because of the outstanding transmission and detection efficiency of the TOF mass analyzer, detection limits in the amol/microl to low fmol/microl range were reached. The usability of LC-ESI-QqTOF-MS for the qualitative and quantitative analysis of synthetic oligonucleotide mixtures was demonstrated.     

Initiation of radical polymerization by peroxyacetates: Polymer end-group analysis by electrospray ionization mass spectrometry

End-groups of poly(methyl methacrylate) from radical solution polymerization of MMA using tert-butyl peroxyacetate (TBPA), tert-amyl peroxyacetate (TAPA), 1,1,2,2- tetramethylpropyl peroxyacetate (TMPPA), and 1,1,3,3-tetramethylbutyl peroxyacetate (TMBPA) as the initiators were analyzed via electrospray ionization mass spectrometry (ESI-MS). The type and the relative concentration of the radical species, which actually initiate macromolecular growth, are determined. In the majority of cases, these species differ from the primary radicals from thermal decomposition of the peroxyacetates. Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was applied for unambiguous peak assignment. The methylcarbonyloxyl radical, which is formed by the decomposition of all peroxyacetates, was found to undergo decarboxylation yielding an initiating methyl radical. TAPA- and TMPPA-derived alkoxyl radicals mainly show β-scission, TMBPA-derived alkoxyl radicals additionally undergo a 1,5-hydrogen-shift reaction. The tert-butoxyl radicals produced from TBPA undergo pronounced chain-transfer reaction prior to their decomposition into methyl radicals and acetone. In the case of using benzene as a relatively inert solvent, the tert-butoxyl radicals exhibit transfer to monomer yielding polymer molecules, which do not carry any initiator-derived end-groups. By using mesitylene as a cosolvent, small amounts of star polymer were generated via multiple hydrogen abstraction by tert-butoxyl radicals from the three individual methyl groups of mesitylene. This uncomplicated procedure of modification of end-group and polymer topology may be attractive for facile adjustment of polymer viscosity in technical processes. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 2453–2467, 2007     

Analytical characterization of microfabricated SU-8 emitters for electrospray ionization mass spectrometry

We present a detailed optimization and characterization of the analytical performance of SU-8-based emitters for electrospray ionization mass spectrometry (ESI/MS). The improved SU-8 fabrication process presented here enhances patterning accuracy and reduces the time and cost of fabrication. All emitters are freestanding and enable sample delivery by both pressure-driven and spontaneous flows. The optimized emitter design incorporates a sharp, double-cantilevered tip implemented to the outlet of an SU-8 microchannel and provides highly sensitive ESI/MS detection. Moreover, the optimized design allows the use of relatively large microchannel dimensions (up to 200 x 50 microm(2), w x h) without sacrificing the detection sensitivity. This is advantageous with a view of preventing emitter clogging and enabling reproducible analysis. The measured limits of detection for the optimized emitter design were 1 nM for verapamil and 4 nM for Glu-fibrinopeptide B with good quantitative linearities between 1 nM and 10 microM (R(2) = 0.9998) for verapamil and between 4 nM and 3 microM (R(2) = 0.9992) for Glu-fibrinopeptide B. The measured tip-to-tip repeatability for signal intensity was 14% relative standard deviation (RSD) (n = 3; 5 microM verapamil) and run-to-run repeatability 4-11% RSD (n = 4; 5 microM verapamil) for all individual emitters tested. In addition, long-term stability of < 2% RSD was maintained for timescales of 30 min even under free flow conditions. SU-8 polymer was also shown to be chemically stable against most of the tested electrospray solvents.     

Characterization of chemically modified steroids for doping control purposes by electrospray ionization tandem mass spectrometry

The discovery of the designer steroid tetrahydrogestrinone (THG) in elite athletes' doping control samples in 2003 demonstrated the availability of steroid derivatives prepared solely for doping purposes. Modern mass spectrometers utilizing electrospray ionization and collisionally activated dissociation (CAD) of analytes allow the structural characterization of steroids and their derivatization sites by the elucidation of fragmentation behaviors. A total of 21 steroids comprising either a 4,9,11-triene, a 3-keto-4-ene or a 3-keto-1-ene nucleus were investigated regarding their dissociation pathways, deuterated analogues were synthesized and fragmentation routes were postulated, permitting the identification of steroidal structures and modifications. Compounds based on a 4,9,11-triene steroid with an ethyl residue at C-13 (gestrinone analogues) generate abundant fragment ions at m/z 241 and 199, whereas the substitution of the C-13 ethyl group by a methyl residue (trenbolone analogues) results in a shift of m/z 241 to 227. Substances related to testosterone with a 3-keto-4-ene structure give rise to abundant fragment ions at m/z 109 and 97 whereas steroids with a 3-keto-1-ene nucleus eliminate the A-ring including the carbons C-1-C-4, in addition to C-19 that is proposed to migrate from C-10 to C-1 under CAD conditions.     

Mass spectrometry in the study of anthocyanins and their derivatives: differentiation of Vitis vinifera and hybrid grapes by liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry

A mass spectrometric-based procedure for anthocyanin profiling was set up to distinguish authentic Vitis vinifera from hybrid red grapevine cultivars. 3-O-Monoglucoside and the related acetyl-, p-coumaryl- and caffeoyl-monoglucoside anthocyanins occurred only in Vitis vinifera, whereas 3,5-O-diglucoside and the substituted acetyl-, p-coumaryl-, feruloyl- and caffeoyl-diglucoside anthocyanins were the additional pigments in hybrid grapevines. The procedure was applied expressly to identify red grape cultivars based on the anthocyanin chemo-type determination. In particular, a red grape cultivar, having 3,5-O-diglucoside anthocyanins and a novel class of anthocyanin monoglucosides, such as cyanidin-3-O-, cyanidin-3-O-(6-O-acetyl)- and cyanidin-3-O-(6-O-p-coumaryl)pentoside, was classified as hybrid. A second vine cultivar, characterized exclusively by 3-O-monoglucoside anthocyanins, was included among the Vitis vinifera species. Anthocyanin profiling by mass spectrometry could represent the core of a chemotaxonomic procedure for distinguishing American and European grapevines based on the identification of post-synthetic anthocyanidin modification.     

Characterization of dinitroporphyrin zinc complexes by electrospray ionization tandem mass spectrometry. Unusual fragmentations of beta-(1,3-dinitroalkyl) porphyrins

The zinc complexes of diaryl bis(p-nitrophenyl)porphyrins and beta-(1,3-dinitroalkyl)tetraphenylporphyrins were studied by electrospray ionization (ESI) tandem mass spectrometry (MS/MS). All porphyrins showed the protonated molecule under ESI conditions. The protonated molecules were induced to fragment and the corresponding ESI tandem mass spectra were analysed. Porphyrins with two p-nitrophenyl groups showed, as expected, characteristic fragmentations including either loss of one nitro group, as the major fragment of the tandem mass spectra, and loss of both nitro groups. In contrast, MS/MS of the beta-(1,3-dinitroalkyl)porphyrins provided interesting and unexpected results such as the absence (or in insignificant abundance) of the ions formed by loss of one nitro group. However, these porphyrins show an abundant fragment due to combined loss of the two nitro groups. Also, the typical beta-cleavage of the alkyl chain is not observed per se, only when combined with loss of HNO2 or *NO2. Instead, alpha-cleavage, with loss of the beta-pyrrolic substituent, is the most favourable process.     

Effect of solution acidity on cytochrome c conformations of alternating current electrospray ionization mass spectrometry

This paper reports notable observations regarding the ion charge states of thermally stable cytochrome c, generated using an alternating current (AC) electrospray ionization (ESI) device. An AC ESI sprayer entrains low-mobility ions to accumulate at the meniscus cone tip prior to the ejection of detached aerosols to produce analyte ions. Therefore, as the solvent acidity varies, protein ions entrained in the AC cone tip are found to change conformation less significantly compared with those in the direct current (DC) cone. We acquired the AC ESI mass spectra of cytochrome c at pH range from 2 to 4. Unlike the DC ESI mass spectra showing clear conformation changes due to denaturing, the AC spectra indicated that only partial denaturing occurs even at extremely acidic pH 2. More native cytochrome c in lower charge states therefore remained. Moreover, with a solvent mixture of aqueous buffer and acetonitrile (70:30), partially denatured cytochrome c was still preserved at pH 2 by using AC ESI. Completely denatured proteins are observed at pH 2 by using DC ESI.     

Field-induced wooden-tip electrospray ionization mass spectrometry for high-throughput analysis of herbal medicines

This study demonstrates the first application of field-induced wooden-tip electrospray ionization (ESI) mass spectrometry (MS) for high-throughput analysis of herbal medicines. By application of an opposite and sample-contactless high voltage on the MS inlet rather than wooden tips, a high-throughput analysis device is easily set up, and a relatively fast analysis speed of 6 s per sample was successfully achieved. In addition, fast polarity switching between positive and negative ion detection mode is readily accomplished, which provides more complete chemical information for quality assessment and control of herbal medicines. By using the proposed method, various active ingredients present in different herbal medicines were rapidly detected, and the obtained mass spectra were served as the samples' fingerprints for tracing the origins, establishing the authenticity, and assessing the quality consistency and stability of herbal medicines. Our experimental results demonstrated that field-induced wooden-tip ESI-MS is a desirable method for high-throughput analysis of herbal medicines, with promising prospects for rapidly differentiating the origin, determining the authenticity, and assessing the overall quality of pharmaceuticals.     

Peptide sequencing through N-terminal phosphonylation and electrospray ionization mass spectrometry

Peptides were phosphonylated at their N-termini by reacting with ethoxyphenylphosphinate in the presence of triethylamine and tetrachloromethane under mild conditions. The phosphonylated peptides were analyzed by tandem electrospray ionization mass spectrometry. N-Terminal phosphonylation selectively increased the intensities of b(n)-type ions relative to other ion types. The resulting simplified mass spectra clearly show the sequential loss of amino acid residues from the C-termini of peptides, providing a convenient and rapid method for peptide sequencing.