Time of flight versus ion trap MS coupled to CE to analyse intact proteins

In this work, two different CE-MS instruments, namely, CE-ESI-IT-MS and CE-ESI-TOF-MS, applied to analyse intact proteins from complex samples are investigated. The aim of this work was to compare both instruments in terms of LOD, number of proteins detected, and precision and repeatability in the determination of the protein relative molecular mass. Results show that although CE-ESI-IT-MS provides cleaner MS spectra of intact proteins, CE-ESI-TOF-MS allows the identification of a higher number of proteins from complex matrices in an easier way. Performance in terms of peak area reproducibility, LOD and precision in the determination of the molecular mass were similar for both instruments. The usefulness of the optimised CE-ESI-IT-MS and CE-ESI-TOF-MS conditions was demonstrated by studying the zein-proteins composition of three natural maize lines and their corresponding transgenic lines, showing no significant differences.     

Critical assessment of ionization patterns and applications of ambient desorption/ionization mass spectrometry using FAPA–MS

Ambient desorption/ionization mass spectrometry (MS) has gained growing interest during the last decade due to its high analytical performance and yet simplicity. Here, one of the recently developed ambient desorption/ionization MS sources, the flowing atmospheric‐pressure afterglow (FAPA) source, was investigated in detail regarding background ions and typical ionization patterns in the positive as well as the negative ion mode for a variety of compound classes, comprising alkanes, alcohols, aldehydes, ketones, carboxylic acids, organic peroxides and alkaloids. A broad range of signals for adducts and losses was found, besides the usually emphasized detection of quasimolecular ions, i.e. [M + H]⁺ and [M ? H]^? in the positive and the negative mode, respectively. It was found that FAPA–MS is best suited for polar analytes containing nitrogen and/or oxygen functionalities, e.g. carboxylic acids, with low molecular weights and relatively high vapor pressures. In addition, the source was used in proof‐of‐principle studies, illustrating the capabilities and limitations of the technique: Firstly, traces of cocaine were detected and unambiguously identified on euro banknotes using FAPA ionization in combination with tandem MS, suggesting a correlation between cocaine abundance and age of the banknote. Secondly, FAPA–MS was used for the identification of acidic marker compounds in organic aerosol samples, indicating yet‐undiscovered matrix and sample surface effects of ionization pathways in the afterglow region. Copyright © 2016 John Wiley & Sons, Ltd.     

An investigation of peak-broadening effects arising when combining CE with MS

In this study, peak-broadening effects caused by nebulizing gas flow and lack of temperature control have been investigated for separation capillaries with three different inner diameters. The study was performed with serial UV/ESI-MS detection in an effort to distinguish between peak broadening arising in the separation and peak broadening arising in the ion source. The nebulizing gas was found to significantly affect both migration time and separation efficiency when using capillaries with 50 and 75 microm id. If the nebulizing gas is on during injection, the injection volume increases to such an extent that significant peak broadening is induced. Reducing the id to 25 microm minimizes the parabolic flow induced by the nebulizing gas. Results indicate that the nebulizing gas pressure can be optimized to minimize peak broadening in the ion source. A decrease in detection sensitivity, possibly related to the orthogonal design of the interface, was observed when the nebulizing gas pressure was increased. A tapered capillary tip was found to provide superior separation efficiency as well as sensitivity.     

CE with on‐line detection by ICP‐MS for studying the competitive binding of zinc against cadmium for glutathione

Development of a feasible method for studying the competitive interaction between a pair of antagonists is essential for understanding the antagonism of trace metals in biological systems. Herein, we report the application of CE on‐line coupled with ICP mass spectroscopy (CE‐ICP‐MS) to investigate the competitive binding of Zn²⁺ against Cd²⁺ for glutathione (GSH), which is related to the detoxification of Cd²⁺ in biological system, and introduce a method to evaluate the kinetics and thermodynamics for the competitive binding of Zn²⁺ against Cd²⁺ for GSH. The CE‐ICP‐MS hybrid technique allows easy and sensitive probing of the competitive binding of Zn²⁺ against Cd²⁺ for GSH and quantitative determination of the important thermodynamic and kinetic parameters of the competitive binding of Zn²⁺ against Cd²⁺ for GSH. Owing to the high sensitivity and element selectivity with multi‐elements detection capacity of ICP‐MS, we detailed the evaluation of the kinetics and thermodynamics describing the competition of Zn²⁺ against Cd²⁺ for GSH from the systematic data obtained by CE‐ICP‐MS. The competitive binding of Zn²⁺ against Cd²⁺ for GSH was demonstrated exothermic and thermodynamically favorable (ΔG=?7.2 kJ/mol) and driven entirely by a large favorable enthalpy decrease (ΔH=?15.1 kJ/mol) but with an unfavorable entropy decrease (ΔS=?25.6 J/mol/K). The kinetic data were fit to a second‐order equation with the reaction rate constant (k) of (2.18±0.10)×10² L/(mol·s) under the simulated physiological condition.     

A simple fabrication method for tapered capillary tip and its applications in high‐speed CE and ESI‐MS

Fabrication of capillaries with tapered tips is an important technique that is required in many analytical chemistry areas, such as ESI‐MS, CE, electrochemical analysis, and microinjection. This paper describes a simple and effective grinding‐based fabrication method for capillaries with tapered tips. A novel grinding mode utilizing the combination of rotation and precession of an elastic capillary was developed, which significantly improved the controllability to the grinding process as well as the capillary tip shape. The capillary was fabricated by fixing it in an electric drill installed perpendicularly, and grind the capillary tip rotated around its own axis as well as the drill axis on sandpapers. Compared with conventional fabrication techniques for capillary tips, the present method is easy to control the capillary tip shape in routine laboratories without the requirement of expensive equipments or poisonous reagent (e.g. hydrofluoric acid (HF) solution). Various capillaries with different tip diameters and tip taper angles could be fabricated using the present method with good controllability and reproducibility. These capillaries were applied in high‐speed CE and ESI‐MS analysis to demonstrate the feasibility and potential of this fabrication method.     

Potential of atmospheric pressure chemical ionization source in GC‐QTOF MS for pesticide residue analysis

The potential applications of a new atmospheric pressure source for GC‐MS analysis have been investigated in this work. A list of around 100 GC‐amenable pesticides, which includes organochlorine, organophosphorus and organonitrogenated compounds, has been used to evaluate their behavior in the new source. Favoring the major formation of the molecular ion in the source has been the main goal due to the wide‐scope screening possibilities that this fact brings in comparison with the traditional, highly fragmented electron ionization spectra. Thus, the addition of water as modifier has been tested as a way to promote the generation of protonated molecules. Pesticides investigated have been classified into six groups according to their ionization/fragmentation behavior. Four of them are characterized by the abundant formation of the protonated molecule in the atmospheric pressure source, mostly being the base peak of the spectrum. These results show that wide‐scope screening could be easily performed with this source by investigating the presence of the protonated molecule ion, MH+. The developed procedure has been applied to pesticide screening in different food samples (nectarine, orange and spinach) and it has allowed the presence of several pesticides to be confirmed such as chlorpyriphos ethyl, deltamethrin and endosulfan sulfate. The availability of a quadrupole time‐of‐flight instrument made it feasible to perform additional MS/MS experiments for both standards and samples to go further in the confirmation of the identity of the detected compounds. Results shown in this paper have been obtained using a prototype source which exhibits promising features that could be applied to other analytical problems apart from those illustrated in this work. Copyright © 2010 John Wiley & Sons, Ltd.     

An overview of LC–MS interfacing systems with selected applications

Advantages and limitations are described for the different LC–MS interfacing systems (moving belt; direct liquid introduction; thermospray; atmospheric pressure ionization with heated pneumatic nebulizer, electrospray, or high flow ion spray; particle beam; and continuous flow fast atom bombardment). Some comments are also made about interfacing capillary zone electrophoresis (CZE). The peculiarities of the various interfaces are described, as are liquid chromatographic requirements prior to mass spectrometry using the different ionization techniques. Selected biological and environmental applications are given.     

Non‐vacuum field desorption ion source implemented under super‐atmospheric pressure

Standard field desorption (FD) ionization is implemented under high vacuum condition. In this paper, non‐vacuum FD is performed under a super‐atmospheric pressure environment using untreated tungsten wires as FD emitter, and the ion source was coupled to a commercial linear ion trap mass spectrometer. The operating pressure of the ion source was 6 bars which was high enough to provide sufficient dielectric strength to the working gas so that the high voltage that was required for FD could be applied to the emitter without occurrence of electrical discharge. Non‐volatile sample deposited on the bare tungsten wire FD emitter was heated by flowing direct current through the emitter. Similar to vacuum FD, the formation of conical protrusion of the liquefied sample layer under the strong electric field was also observed. Using the present ion source, high pressure field‐desorption of polar neutral compounds, organic salts and ionic liquids is demonstrated. Copyright © 2012 John Wiley & Sons, Ltd.     

HPLC‐PAD‐atmospheric pressure chemical ionization‐MS metabolite profiling of cytotoxic carotenoids from the echinoderm Marthasterias glacialis (spiny sea‐star)

An HPLC‐PAD‐atmospheric pressure chemical ionization‐MS metabolite profiling analysis was conducted on the marine echinoderm Marthasterias glacialis (spiny sea‐star). Bio‐guided purification of the methanolic extract led to the isolation of several carotenoids, namely zeaxanthin, astaxanthin and lutein. These compounds were characterized using both UV–Vis characteristics and MS spectra interpretation. No previous works addressed the MS analysis of carotenoids present in this organism. The purified carotenoid fraction displayed a strong cell proliferation inhibition against rat basophilic leukemia RBL‐2H3 (IC₂₅=268 μg/mL) cancer cell line. Against healthy V79 (rat lung fibroblasts (IC₂₅=411 μg/mL)) cell line, however, toxicity was lower, as it is desired for anti‐cancer molecules. This study suggests that M. glacialis may constitute a good source of bioactive compounds that can be used as lead compounds for the pharmaceutical industry.     

Identification of polyimide materials using quantitative CE with UV and QTOF‐MS detection

Polyimides (PIs) are a group of widely used synthetic materials that service a variety of different purposes including microelectronics, insulating films and aerospace applications. Depending on the requirements (defined by the particular final product), the actual composition of PIs may show substantial chemical variation. To study this variation in chemical structure, CE‐MS can be employed for the determination of PI composition following chemical degradation of the polymer sample. PI is chemically decomposed to corresponding aromatic diamine and carboxylic acid components using an alkali fusion reaction. Solid polymer samples are fused in a potassium hydroxide melt yielding reaction products that are diluted in acid and can be immediately analysed by CE coupled to a Q/TOF‐MS with quantification performed using conventional UV detection. This approach involves a simple and rapid sample preparation yielding both qualitative and quantitative information regarding the chemical composition of the polymer. Application of the CE‐MS approach is shown for a range of commercially available PI and poly(amide–imide) materials and the results are used to infer the respective chemical compositions.     

Study of different atmospheric-pressure interfaces for LC-MS/MS determination of acrylamide in water at sub-ppb levels

A rapid, sensitive and selective method based on LC-MS/MS has been developed for the direct determination of acrylamide residues in water in compliance with the current European Union (EU) 98/83 Drinking Water Directive. Given the high polarity of acrylamide, the application of a rapid on-line solid phase extraction step, commonly used for preconcentrating low analyte levels, was not found to be completely satisfactory. Therefore, an alternative approach based on the use of direct large-volume injection into the LC-MS/MS system has been used. Three atmospheric-pressure interfaces (ESI, APCI and Ion Sabre APCI) were checked to reach the required sensitivity (0.1 microg/l). All three interfaces were tested by analysis of six different water samples (surface water, groundwater, drinking water and three treated water samples) spiked at three concentration levels each (0.1, 1 and 10 microg/l). When using ESI, poor sensitivity and high matrix effects were observed. This situation improved when APCI was used as the interface because no matrix effect was found, although sensitivity was not completely satisfactory. The best results were obtained by interfacing the Ion Sabre APCI; its higher sensitivity for acrylamide (LOD 0.03 microg/l) and the absence of matrix effects recommended its selection. Using this approach, satisfactory recoveries (90-97%) and precision (<12%) were obtained for all water samples studied. Besides, the acquisition of two different MS/MS transitions allowed not only the quantification but also the confirmation of acrylamide in water at concentration levels around 0.1 microg/l.     

Atmospheric pressure chemical ionization of explosives using alternating current corona discharge ion source

The high‐sensitive detection of explosives is of great importance for social security and safety. In this work, the ion source for atmospheric pressure chemical ionization/mass spectrometry using alternating current corona discharge was newly designed for the analysis of explosives. An electromolded fine capillary with 115 µm inner diameter and 12 mm long was used for the inlet of the mass spectrometer. The flow rate of air through this capillary was 41 ml/min. Stable corona discharge could be maintained with the position of the discharge needle tip as close as 1 mm to the inlet capillary without causing the arc discharge. Explosives dissolved in 0.5 µl methanol were injected to the ion source. The limits of detection for five explosives with 50 pg or lower were achieved. In the ion/molecule reactions of trinitrotoluene (TNT), the discharge products of NO_x^? (x = 2,3), O₃ and HNO₃ originating from plasma‐excited air were suggested to contribute to the formation of [TNT ? H]^? (m/z 226), [TNT ? NO]^? (m/z 197) and [TNT ? NO + HNO₃]^? (m/z 260), respectively. Formation processes of these ions were traced by density functional theory calculations. Copyright © 2015 John Wiley & Sons, Ltd.     

CE‐MS with electrokinetic supercharging and application to determination of neurotransmitters

Electrokinetic supercharging (EKS) is known as one of the most effective online electrophoretic preconcentration techniques, though pairing with it with mass spectrometry has presented challenges. Here, EKS is successfully paired with ESI‐MS/MS to provide a sensitive and robust method for analysis of biogenic amines in biological samples. Injection parameters including electric field strength and the buffer compositions used for the separation and focusing were investigated to achieve suitable resolution, high sensitivity, and compatibility with ESI‐MS. Using EKS, the sensitivity of the method was improved 5000‐fold compared to a conventional hydrodynamic injection with CZE. The separation allowed for baseline resolution of several neurotransmitters within 16 min with LODs down to 10 pM. This method was applied to targeted analysis of seven biogenic amines from rat brain stem and whole Drosophila tissue. This is the first method to use EKS with CE‐ESI‐MS/MS to analyze biological samples.     

Determination of peptide hormones of brain and intestine by CE with ESI-MS detection

A new method for the determination of the peptide hormones of brain and intestine based on CE coupling with a DAD and ESI-MS was established. Several electrophoretic and ESI-MS parameters were investigated in detail, such as electrolyte nature and concentration, organic solvent and sheath liquid compositions, nebulization gas pressure and the ESI capillary voltage. Optimized conditions were achieved with 25 mM formic acid-ammonium formate (pH 2.9) as the optimal electrolyte, 2 mM formic acid in 80% methanol in water as the sheath liquid, and 20 kV applied voltage. Under the optimized conditions, four protonated peptides were separated by CE and selectively detected by a quadrupole mass spectrometer with a sheath flow ESI interface. LODs for the four peptides (neurotensin hexapeptide, neurotensin, cholecystokinin tetrapeptide, and pentagastrin) were in the range of 0.10-0.60 micromol/L at an S/N of 3. The RSDs (n = 8) of the method were 0.70-1.5% for migration times and 1.6-6.1% for peak areas. This method is simple, rapid, and selective compared with RIA and ELISA techniques, and has been applied to the analysis of rat hypothalamus tissue.     

Analysis of impurities in pharmaceuticals by LC‐MS with cold electron ionization

Pharmaceuticals require careful and precise determination of their impurities that might harm the user upon consumption. Although today, the most common technique for impurities identification is liquid chromatography‐mass spectrometry (LC‐MS/MS), it has several downsides due to the nature of the ionization method. Also, the analyses in many cases are targeted thus despite being present, some of the compounds will not be revealed. In this paper, we propose and show a new method for untargeted analysis and identification of impurities in active pharmaceutical ingredients (APIs). The instrument used for these analyses is a novel electron ionization (EI) LC‐MS with supersonic molecular beams (SMB). The EI‐LC‐MS‐SMB was implemented for analyses of several drug samples spiked with an impurity. The instrument provides EI mass spectra with enhanced molecular ions, named Cold EI, which increases the identification probabilities when the compound is identified with the aid of an EI library like National Institute of Standards and Technology (NIST). We analyzed ibuprofen and its impurities, and both the API and the expected impurity were identified with names and structures by the NIST library. Moreover, other unexpected impurities were found and identified proving the ability of the EI‐LC‐MS‐SMB system for truly untargeted analysis. The results show a broad dynamic range of four orders of magnitude at the same run with a signal‐to‐noise ratio of over 10 000 for the API and almost uniform response.     

Highly sensitive detection of five typical fluoroquinolones in low‐fat milk by field‐enhanced sample injection‐based CE in bubble cell capillary

Fluoroquinolones are a group of synthetic antibiotics with a broad activity spectrum against mycoplasma, Gram‐positive, and Gram‐negative bacteria. Due to the extensive use of fluoroquinolones in farming and veterinary science, there is a constant need in the analytical methods able to efficiently monitor their residues in food products of animal origin, regulated by Commission Regulation (European Union) no. 37/2010. Herein, field‐enhanced sample injection for sample stacking prior the CZE separation was developed inside a bubble cell capillary for highly sensitive detection of five typical fluoroquinolones in bovine milk. Ethylenediamine was proposed as the main component of BGE for the antibiotics separation. The effect of BGE composition, injection parameters, and water plug length on the field‐enhanced sample injection‐based CE with UV detection was investigated. Under the optimized conditions, described field‐enhanced sample injection‐based CE‐UV analysis of fluoroquinolones provides LODs varying from 0.4 to 1.3 ng/mL. These LOD values are much lower (from 460 to 1500 times) than those obtained by a conventional CE in a standard capillary without bubble cell. The developed method was finally applied for the analysis of fluoroquinolones in low‐fat milk from a Swiss supermarket. Sample recovery values from 93.6 to 106.0% for different fluoroquinolones, and LODs from 0.7 to 2.5 μg/kg, were achieved. Moreover, the proposed ethylenediamine‐based BGE as volatile and compatible with MS system, enabled the coupling of the field‐enhanced sample injection‐based CE with a recently introduced electrostatic spray ionization MS via an iontophoretic fraction collection interface for qualitative fluoroquinolones identification.     

APCI as an innovative ionization mode compared with EI and CI for the analysis of a large range of organophosphate esters using GC‐MS/MS

Organophosphate esters (OPEs) are chemical compounds incorporated into materials as flame‐proof and/or plasticizing agents. In this work, 13 non‐halogenated and 5 halogenated OPEs were studied. Their mass spectra were interpreted and compared in terms of fragmentation patterns and dominant ions via various ionization techniques [electron ionization (EI) and chemical ionization (CI) under vacuum and corona discharge atmospheric pressure chemical ionization (APCI)] on gas chromatography coupled to mass spectrometry (GC‐MS). The novelty of this paper relies on the investigation of APCI technique for the analysis of OPEs via favored protonation mechanism, where the mass spectra were mostly dominated by the quasi‐molecular ion [M + H]⁺. The EI mass spectra were dominated by ions such as [H₄PO₄]⁺, [M–R]⁺, [M–Cl]⁺, and [M–Br]⁺, and for some non‐halogenated aryl OPEs, [M]^+● was also observed. The CI mass spectra in positive mode were dominated by [M + H]⁺ and sometimes by [M–R]⁺, while in negative mode, [M–R]⁻ and more particularly [X]^‐ and [X₂]^‐● were mainly observed for the halogenated OPEs. Both EI and APCI techniques showed promising results for further development of instrumental method operating in selective reaction monitoring mode. Instrumental detection limits by using APCI mode were 2.5 to 25 times lower than using EI mode for the non‐brominated OPEs, while they were determined at 50‐100 times lower by the APCI mode than by the EI mode, for the two brominated OPEs. The method was applied to fish samples, and monitored transitions by using APCI mode showed higher specificity but lower stability compared with EI mode. The sensitivity in terms of signal‐to‐noise ratio varying from one compound to another. Copyright © 2016 John Wiley & Sons, Ltd.     

Analysis of lipids with desorption atmospheric pressure photoionization‐mass spectrometry (DAPPI‐MS) and desorption electrospray ionization‐mass spectrometry (DESI‐MS)

In this article, the effect of spray solvent on the analysis of selected lipids including fatty acids, fat‐soluble vitamins, triacylglycerols, steroids, phospholipids, and sphingolipids has been studied by two different ambient mass spectrometry (MS) methods, desorption electrospray ionization‐MS (DESI‐MS) and desorption atmospheric pressure photoionization‐MS (DAPPI‐MS). The ionization of the lipids with DESI and DAPPI was strongly dependent on the spray solvent. In most cases, the lipids were detected as protonated or deprotonated molecules; however, other ions were also formed, such as adduct ions (in DESI), [M‐H]⁺ ions (in DESI and DAPPI), radical ions (in DAPPI), and abundant oxidation products (in DESI and DAPPI). DAPPI provided efficient desorption and ionization for neutral and less polar as well as for ionic lipids but caused extensive fragmentation for larger and more labile compounds because of a thermal desorption process. DESI was more suitable for the analysis of the large and labile lipids, but the ionization efficiency for less polar lipids was poor. Both methods were successfully applied to the direct analysis of lipids from pharmaceutical and food products. Although DESI and DAPPI provide efficient analysis of lipids, the multiple and largely unpredictable ionization reactions may set challenges for routine lipid analysis with these methods. Copyright © 2012 John Wiley & Sons, Ltd.     

Potential of prodendronic polyamines with modulated segmental charge density as novel coating for fast and efficient analysis of peptides and basic proteins by CE and CE‐MS

In this work, the suitability of a new polymer family has been investigated as capillary coatings for the analysis of peptides and basic proteins by CE. This polymer family has been designed to minimize or completely prevent protein–capillary wall interactions and to modify the EOF. These coating materials are linear polymeric chains bearing as side cationizable moiety a dentronic triamine derived from N,N,N’,N’‐tetraethyldiethylenetriamine (TEDETA), which is linked to the backbone through a spacer (unit labeled as TEDETAMA). Four different polymers have been prepared and evaluated: a homopolymer which comprised only of those cationizable repetitive units of TEDETAMA, and three copolymers that randomly incorporate TEDETAMA together with neutral hydrosoluble units of N‐(2‐hydroxypropyl) methacrylamide (HPMA) at different molar percentages (25:75, 50:50 and 75:25). It has been demonstrated that the composition of the copolymers influences the EOF and therefore the separation of the investigated biopolymers. Among the novel polymers studied, poly‐(TEDETAMA‐co‐HPMA) 50:50 copolymer was successfully applied as coating material of the inner capillary surface in CE‐UV and CE‐MS, providing EOF reversing together with fast and efficient baseline separation of peptides and basic proteins. Finally, the feasibility of the polymer‐coated capillary was shown through the analysis of lysozyme in a cheese sample.     

Efficient approach for the detection and identification of new androgenic metabolites by applying SRM GC‐CI‐MS/MS: a methandienone case study

Identification of anabolic androgenic steroids (AAS) is a vital issue in doping control and toxicology, and searching for metabolites with longer detection times remains an important task. Recently, a gas chromatography chemical ionization triple quadrupole mass spectrometry (GC‐CI‐MS/MS) method was introduced, and CI, in comparison with electron ionization (EI), proved to be capable of increasing the sensitivity significantly. In addition, correlations between AAS structure and fragmentation behavior could be revealed. This enables the search for previously unknown but expected metabolites by selection of their predicted transitions. The combination of both factors allows the setup of an efficient approach to search for new metabolites. The approach uses selected reaction monitoring which is inherently more sensitive than full scan or precursor ion scan. Additionally, structural information obtained from the structure specific CI fragmentation pattern facilitates metabolite identification. The procedure was demonstrated by a methandienone case study. Its metabolites have been studied extensively in the past, and this allowed an adequate evaluation of the efficiency of the approach. Thirty three metabolites were detected, including all relevant previously discovered metabolites. In our study, the previously reported long‐term metabolite (18‐nor‐17β‐hydroxymethyl,17α‐methyl‐androst‐1,4,13‐trien‐3‐one) could be detected up to 26 days by using GC‐CI‐MS/MS. The study proves the validity of the approach to search for metabolites of new synthetic AAS and new long‐term metabolites of less studied AAS and illustrates the increase in sensitivity by using CI. Copyright © 2016 John Wiley & Sons, Ltd.