Plasma-Assisted Reaction Chemical Ionization for Elemental Mass Spectrometry of Organohalogens

We present plasma-assisted reaction chemical ionization (PARCI) for elemental analysis of halogens in organic compounds. Organohalogens are broken down to simple halogen-containing molecules (e.g., HBr) in a helium microwave-induced plasma followed by negative mode chemical ionization (CI) in the afterglow region. The reagent ions for CI originate from penning ionization of gases (e.g., N₂) introduced into the afterglow region. The performance of PARCI-mass spectrometry (MS) is evaluated using flow injection analyses of organobromines, demonstrating 5–8 times better sensitivities compared with inductively coupled plasma MS. We show that compound-dependent sensitivities in PARCI-MS mainly arise from sample introduction biases.

Minimization of Fragmentation and Aggregation by Laser Desorption Laser Ionization Mass Spectrometry

Measuring average quantities in complex mixtures can be challenging for mass spectrometry, as it requires ionization and detection with nearly equivalent cross-section for all components, minimal matrix effect, and suppressed signal from fragments and aggregates. Fragments and aggregates are particularly troublesome for complex mixtures, where they can be incorrectly assigned as parent ions. Here we study fragmentation and aggregation in six aromatic model compounds as well as petroleum asphaltenes (a naturally occurring complex mixture) using two laser-based ionization techniques: surface assisted laser desorption ionization (SALDI), in which a single laser desorbs and ionizes solid analytes; and laser ionization laser desorption mass spectrometry (L²MS), in which desorption and ionization are separated spatially and temporally with independent lasers. Model compounds studied include molecules commonly used as matrices in single laser ionization techniques such as matrix assisted laser desorption ionization (MALDI). We find significant fragmentation and aggregation in SALDI, such that individual fragment and aggregate peaks are typically more intense than the parent peak. These fragment and aggregate peaks are expected in MALDI experiments employing these compounds as matrices. On the other hand, we observe no aggregation and only minimal fragmentation in L²MS. These results highlight some advantages of L²MS for analysis of complex mixtures such as asphaltenes.

Multidimensional nano-HPLC coupled with tandem mass spectrometry for analyzing biotinylated proteins

Multidimensional high-performance liquid chromatography (HPLC) is a key method in shotgun proteomics approaches for analyzing highly complex protein mixtures by complementary chromatographic separation principles. Here, we describe an integrated 3D-nano-HPLC/nano-electrospray ionization quadrupole time-of-flight mass spectrometry system that allows an enzymatic digestion of proteins followed by an enrichment and subsequent separation of the created peptide mixtures. The online 3D-nano-HPLC system is composed of a monolithic trypsin reactor in the first dimension, a monolithic affinity column with immobilized monomeric avidin in the second dimension, and a reversed phase C18 HPLC-Chip in the third dimension that is coupled to a nano-ESI-Q-TOF mass spectrometer. The 3D-LC/MS setup is exemplified for the identification of biotinylated proteins from a simple protein mixture. Additionally, we describe an online 2D-nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS setup for the enrichment, separation, and identification of cross-linked, biotinylated species from chemical cross-linking of cytochrome c and a calmodulin/peptide complex using a novel trifunctional cross-linker with two amine-reactive groups and a biotin label.

Imaging of Noncovalent Complexes by MALDI-MS

Noncovalent interactions govern how molecules communicate. Mass spectrometry is an important and versatile tool for the analysis of noncovalent complexes (NCX). Electrospray mass spectrometry (ESI-MS) is the most widely used MS technique for the study of NCXs because of its softer ionization and easy compatibility with the solution phase of NCX mixtures. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has also been used to study NCXs. However, successful analysis depends upon several experimental factors, such as matrix selection, solution pH, and instrumental parameters. In this study, we employ MALDI imaging mass spectrometry to investigate the location and formation of NCXs, involving both peptides and proteins, in a MALDI sample spot.

Simultaneous serum desalting and total protein determination by macroporous reversed-phase chromatography

Macroporous reversed-phase (mRP) chromatography was successfully used to develop an accurate and precise method for total protein in serum. The limits of detection (0.83 μg, LOD) and quantification (2.51 μg, LOQ) for the mRP method are comparable with those of the widely used micro BCA protein assay. The mRP method can be used to determine the total protein concentration across a wide dynamic range by detecting chromatographic peaks at 215 nm and 280 nm. The method has the added advantage of desalting and denaturing proteins, leading to more complete digestion by trypsin and to better LC–MS–MS identification in shotgun proteomics experiments.

Direct Analysis of Triacylglycerols from Crude Lipid Mixtures by Gold Nanoparticle-Assisted Laser Desorption/Ionization Mass Spectrometry

Triacylglycerols (TAGs), essential energy storage lipids, are easily detected by conventional MALDI MS when occurring on their own. However, their signals are easily overwhelmed by other lipids, mainly phosphatidylcholines (PCs) and, therefore, require purification. In order to profile TAGs from crude lipid mixtures without prefractionation, we investigated alternative matrixes that can suppress phospholipid ion signals and enhance cationization of TAGs. We found that an aqueous solution of citrate-capped gold nanoparticles (AuNPs) with a diameter of 12 nm is a superior matrix for the laser desorption/ionization mass spectrometry (LDI MS) of TAGs in crude lipid mixtures. The AuNP matrix effectively suppressed other lipid signals such as phospholipids and also provided 100 times lower detection limit for TAGs than 2,5-dihydroxybenzoic acid (DHB), the best conventional MALDI matrix for TAGs. The AuNP-assisted LDI MS enabled us to obtain detailed TAG profiles including minor species directly from crude beef lipid extracts without phospholipid interference. In addition, we could detect TAGs at a trace level from a total brain lipid extract.

Analysis of Biomolecules by Atmospheric Pressure Visible-Wavelength MALDI-Ion Trap-MS in Transmission Geometry

We report the development of a new AP visible-wavelength MALDI-ion trap-MS instrument with significantly improved performance over our previously reported system (Int. J. Mass Spectrom. 315, 66–73 (2012)). A Nd:YAG pulsed laser emitting light at 532 nm was used to desorb and ionize oligosaccharides and peptides in transmission geometry through a glass slide. Limits of detection (LODs) achieved in MS mode correspond to picomole quantities of oligosaccharides and femtomole quantities of peptides. Tandem MS (MS/MS) experiments enabled identification of enzymatically digested proteins and oligosaccharides by comparison of MS/MS spectra with data found in protein and glycan databases. Moreover, the softness of ionization, LODs, and fragmentation spectra of biomolecules by AP visible-wavelength MALDI-MS were compared to those obtained by AP UV MALDI-MS using a Nd:YAG laser emitting light at 355 nm. AP visible-wavelength MALDI appears to be a softer ionization technique then AP UV MALDI for the analysis of sulfated peptides, while visible-wavelength MALDI-MS, MS/MS, and MS/MS/MS spectra of other biomolecules analyzed were mostly similar to those obtained by AP UV MALDI-MS. Therefore, the methodology presented will be useful for MS and MSⁿ analyses of biomolecules at atmospheric pressure. Additionally, the AP visible-wavelength MALDI developed can be readily used for soft ionization of analytes on various mass spectrometers.

Data-Independent Microbial Metabolomics with Ambient Ionization Mass Spectrometry

Atmospheric ionization methods are ideally suited for prolonged MS/MS analysis. Data-independent MS/MS is a complementary technique for analysis of biological samples as compared to data-dependent analysis. Here, we pair data-independent MS/MS with the ambient ionization method nanospray desorption electrospray ionization (nanoDESI) for untargeted analysis of bacterial metabolites. Proof-of-principle data and analysis are illustrated by sampling Bacillus subtilis and Pseudomonas aeruginosa directly from Petri dishes. We found that this technique enables facile comparisons between strains via MS and MS/MS plots which can be translated to chemically informative molecular maps through MS/MS networking. The development of novel techniques to characterize microbial metabolites allows rapid and efficient analysis of metabolic exchange factors. This is motivated by our desire to develop novel techniques to explore the role of interspecies interactions in the environment, health, and disease. This is a contribution to honor Professor Catherine C. Fenselau in receiving the prestigious ASMS Award for a Distinguished Contribution in Mass Spectrometry for her pioneering work on microbial mass spectrometry.

p-Tolyl isocyanate derivatization for analysis of CWC-related polar degradation products by mass spectrometry

Most of the precursors and/or degradation products related to the Chemical Weapons Convention (CWC) are polar. Identification of these molecules in environmental samples provides clues regarding the alleged usage and/or synthesis of the parent toxic chemicals. Such polar compounds need to be derivatized in order to analyze them by gas chromatography–mass spectrometry (GC–MS). In this study, we developed a new derivatizing reagent, para-tolyl isocyanate (PTI), for derivatization of polar CWC-related compounds. The PTI reagent selectively derivatizes the –OH and/or?SH functional groups with high efficiency, but does not react with carboxylic acid (?COOH) or phosphonic acid (?(O)P(OH)₂) groups. The PTI derivatives of dialkyl aminoethanols, dialkyl aminoethanol-N-oxides, and 3-quinuclidinol were successfully eluted through GC, and their electron ionization (EI) mass spectra were distinct and provided the structure information by which the isomeric compounds can be easily distinguished. We also calculated the GC-retention index values that can be used for further confirmation of the target compounds. All the studied PTI derivatives can be analyzed by EI-MS with direct insertion probe and/or by direct electrospray ionization mass spectrometry (ESI-MS) together with the MS–MS data; both sets of data provide full structure information. The PTI reagent was found to be better in some respects than the conventional bistrimethylsilyl trifluoroacetamide (BSTFA), a trimethyl silylating reagent. The PTI reagent is commercially available, and the PTI derivatives are highly stable for months and are not sensitive to moisture. The applicability of the PTI derivatization for trace-level determination of the target CWC-related polar compounds in environmental matrices and in human plasma samples is also evaluated.

Laser-Induced Hydrogen Radical Removal in UV MALDI-MS Allows for the Differentiation of Flavonoid Monoglycoside Isomers

Negative-ion matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectra and tandem mass spectra of flavonoid mono-O-glycosides showed the irregular signals that were 1 and/or 2 Da smaller than the parent deprotonated molecules ([M – H]^–) and the sugar-unit lost fragment ions ([M – Sugar – H]^–). The 1 and/or 2 Da mass shifts are generated with the removing of a neutral hydrogen radical (H*), and/or with the homolytic cleavage of the glycosidic bond, such as [M – H* – H]^–, [M – Sugar – H* – H]^–, and [M – Sugar – 2H* – H]^–. It was revealed that the hydrogen radical removes from the phenolic hydroxy groups on the flavonoids, not from the sugar moiety, because the flavonoid backbones themselves absorb the laser. The glycosyl positions depend on the extent of the hydrogen radical removals and that of the homolytic cleavage of the glycosidic bonds. Flavonoid mono-glycoside isomers were distinguished according to their TOF MS and tandem mass spectra.

Mass Discrimination in High-Mass MALDI-MS

In high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), the accessible m/z range is limited by the detector used. Therefore, special high-mass detectors based on ion conversion dynodes (ICDs) have been developed. Recently, we have found that mass bias may exist when such ICD detectors are used [Weidmann et al., Anal. Chem. 85(6), 3425–3432 (2013)]. In this contribution, the mass-dependent response of an ICD detector was systematically studied, the response factors for proteins with molecular weights from 35.9 to 129.9 kDa were determined, and the reasons for mass bias were identified. Compared with commonly employed microchannel plate detectors, we found that the mass discrimination is less pronounced, although ions with higher masses are weakly favored when using an ICD detector. The relative response was found to depend on the laser power used for MALDI; low-mass ions are discriminated against with higher laser power. The effect of mutual ion suppression in dependence of the proteins used and their molar ratio is shown. Mixtures consisting of protein oligomers that only differ in mass show less mass discrimination than mixtures consisting of different proteins with similar masses. Furthermore, mass discrimination increases for molar ratios far from 1. Finally, we present clear guidelines that help to choose the experimental parameters such that the response measured matches the actual molar fraction as closely as possible.

Using electrospray laser desorption ionization mass spectrometry to rapidly examine the integrity of proteins stored in various solutions

Electrospray laser desorption ionization mass spectrometry (ELDI/MS) allows the rapid desorption and ionization of proteins from solutions under ambient conditions. In this study, we have demonstrated the use of ELDI/MS to efficiently examine the integrity of the proteins stored in various solutions before they were further used for other biochemical tests. The protein standards were prepared in the solutions containing buffers, organic salts, inorganic salts, strong acid, strong base, and organic solvents, respectively, to simulate those collected from solvent extraction, filtration, dialysis, or chromatographic separation. Other than the deposit of a drop of the sample solution on the metallic sample plate in an ELDI source, no additional sample pretreatment is needed. The sample drop was then irradiated with a pulsed laser; this led to desorption of the analyte molecules, which subsequently entered the ESI plume to undergo post-ionization. Because adjustment of the composition of the sample solution is unnecessary, this technique appears to be useful for rapidly evaluating the integrity of proteins after storage or prior to further biochemical treatment. In addition, when using acid-free and low-organic-solvent ESI solutions for ELDI/MS analysis, the native conformations of the proteins in solution could be detected.

Resonance-enhanced multiphoton ionization with circularly polarized light: chiral carbonyls

An introduction to the principle and possibilities of the new method of circular dichroism laser mass spectrometry is given and its state of development is reviewed. This method allows enantiosensitive, mass-selective probing of chiral molecules. It is based on the combination of resonance-enhanced multiphoton ionization with circularly polarized light and specially modified time-of-flight mass spectrometry. As an example, application to carbonyls is presented.

Flow injection of liquid samples to a mass spectrometer with ionization under vacuum conditions: a combined ion source for single-photon and electron impact ionization

Electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo-ionization (APPI) are the most important techniques for the ionization of liquid samples. However, working under atmospheric pressure conditions, all these techniques involve some chemical rather than purely physical processes, and therefore, side reactions often yield to matrix-dependent ionization efficiencies. Here, a system is presented that combines both soft single-photon ionization (SPI) and hard 70 eV electron impact ionization (EI) of dissolved compounds under vacuum conditions. A quadrupole mass spectrometer was modified to enable direct EI, a technique developed by Cappiello et al. to obtain library-searchable EI mass spectra as well as soft SPI mass spectra of sample solutions. An electron beam-pumped rare gas excimer lamp working at 126 nm was used as well as a focusable vacuum UV light source for single-photon ionization. Both techniques, EI and SPI, were applied successfully for flow injection experiments providing library-matchable EI fragment mass spectra and soft SPI mass spectra, showing dominant signals for the molecular ion. Four model compounds were analyzed: hexadecane, propofol, chlorpropham, and eugenol, with detection limits in the picomolar range. This novel combination of EI and SPI promises great analytical benefits, thanks to the possibility of combining database alignment for EI data and molecular mass information provided by SPI. Possible applications for the presented ionization technology system are a matrix-effect-free detection and a rapid screening of different complex mixtures without time-consuming sample preparation or separation techniques (e.g., for analysis of reaction solutions in combinatorial chemistry) or a switchable hard (EI) and soft (SPI) MS method as detection step for liquid chromatography.

Study of the ESI and APCI interfaces for the UPLC–MS/MS analysis of pesticides in traditional Chinese herbal medicine

In this work, 53 selected pesticides of different chemical groups were extracted from Chinese herbal medicines and determined by ultra-high-performance liquid chromatography (UHPLC)–tandem mass spectrometry (MS/MS) using both electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI). Extracts were obtained using the acetonitrile-based quick, easy, cheap, effective, rugged, and safe (QuEChERS) sample preparation technique. Cleanup was performed by dispersive solid-phase extraction using primary secondary amine, graphitized carbon black, and octadecylsilane. Two atmospheric-pressure interfaces, ESI and APCI, were checked and compared. The validation study, including detection limits, linearity, and matrix effects, was conducted on fritillaria, radix ginseng, folium isatidis, semen persicae, and flos lonicerae in multiple reaction monitoring mode. These matrices represent a variety of plants used in traditional Chinese medicine. Fritillaria and radix ginseng were chosen as representatives for roots, folium isatidis was chosen as a representative for leaves, semen persicae was chosen as a representative for seeds, and flos lonicerae was chosen as a representative for flowers. The limits of detection for pesticides were lower in the UHPLC–ESI-MS/MS method than in the UHPLC–APCI-MS/MS method. Matrix effects on the two ionizations were evaluated for the five matrices. Soft signal enhancement in UHPLC–APCI-MS/MS and signal suppression in UHPLC–ESI-MS/MS were observed.

Development of liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry for analysis of halogenated flame retardants in wastewater

Until recently, atmospheric pressure photoionization (APPI) has typically been used for the determination of non-polar halogenated flame retardants (HFRs) by liquid chromatography (LC) tandem mass spectrometry. In this study, we demonstrated the feasibility of utilizing liquid chromatography atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (LC-APCI-MS/MS) for analysis of 38 HFRs. This developed method offered three advantages: simplicity, rapidity, and high sensitivity. Compared with APPI, APCI does not require a UV lamp and a dopant reagent to assist atmospheric pressure ionization. All the isomers and the isobaric compounds were well resolved within 14-min LC separation time. Excellent instrument detection limits (6.1 pg on average with 2.0 μL injection) were observed. The APCI mechanism was also investigated. The method developed has been applied to the screening of wastewater samples for screening purpose, with concentrations determined by LC-APCI-MS/MS agreeing with data obtained via gas chromatography high resolution mass spectrometry.

Quantification of coumarin in cinnamon and woodruff beverages using DIP-APCI-MS and LC-MS

The use of the direct inlet probe–atmospheric-pressure chemical ionization (DIP-APCI) ion source developed in our laboratory coupled to a high resolution Q-TOF MS for the quantitative analysis of coumarin in different cinnamon samples was demonstrated in this study. Extraction of coumarin from various cinnamon samples was followed by DIP-APCI-mass spectrometry (MS) and liquid chromatography (LC)-MS analysis. For quantification, an external calibration with and without the use of stable isotope-labeled coumarin as internal standard was compared. The results obtained by DIP-APCI-MS and LC-MS were in good agreement. Even without the use of an internal standard satisfying linearity (R ²?>?0.997), recovery (94–104 % for spiking levels between 100 and 5,000 mg/kg) and intra- and interday repeatability (2.2–13.8 %RSD) was demonstrated using DIP-APCI-MS. To reduce the number of samples requiring quantitative analysis, the possibility of semi-quantitative screening of coumarin directly from powdered cinnamon using DIP-APCI-MS was shown. The analysis of woodruff-flavored beverages and cinnamon-flavored chewing gum by DIP-APCI-MS resulted in the formation of an artifact interfering with coumarin detection. As with other ambient ionization methods, special attention has to be paid to possible spectral interferences due to isobaric substances present in the sample matrix or formed from matrix components after ionization. The temperature-programmed vaporization in DIP-APCI-MS combined with the use of stable isotope-labeled coumarin as internal standard helped in recognizing this interference.

Rapid comprehensive characterization of crude oils by thermogravimetry coupled to fast modulated gas chromatography–single photon ionization time-of-flight mass spectrometry

Comprehensive multi-dimensional hyphenation of a thermogravimetry device (i.e. a thermobalance) to gas chromatography and single photon ionization–time-of-flight mass spectrometry (TG–GC×SPI–MS) has been used to investigate two crude oil samples of different geographical origin. The source of the applied vacuum ultraviolet radiation is an electron beam pumped rare gas excimer lamp (EBEL). The soft photoionization favors the formation of molecular ions. Introduction of a fast, rapidly modulated gas chromatographic separation step in comparison with solely TG–SPI–MS enables strongly enhanced detection especially with such highly complex organic matrices as crude oil. In contrast with former TG–SPI–MS measurements, separation and identification of overlying substances is possible because of different GC retention times. The specific contribution of isobaric compounds to one mass signal is determined for alkanes, naphthalenes, alkylated benzenes, and other compounds.

Use of Doubly Charged Precursors to Validate Dissociation Mechanisms of Singly Charged Poly(Dimethylsiloxane) Oligomers

Collision-induced dissociation of doubly charged poly(dimethylsiloxane) (PDMS) molecules was investigated to provide experimental evidence for fragmentation reactions proposed to occur upon activation of singly charged oligomers. This study focuses on two PDMS species holding trimethylsilyl or methoxy end-groups and cationized with ammonium. In both cases, introduction of the additional charge did not induce significant differences in dissociation behavior, and the use of doubly charged precursors enabled the occurrence of charge-separation reactions, allowing molecules always eliminated as neutrals upon activation of singly charged oligomers to be detected as cationized species. In the case of trimethylsilyl-terminated oligomers, random location of the adducted charge combined with rapid consecutive reactions proposed to occur from singly charged precursors could be validated based on MS/MS data of doubly charged oligomers. In the case of methoxy-terminated PDMS, favored interaction of the adducted ammonium with both end-groups, proposed to rationalize the dissociation behavior of singly charged molecules, was also supported by MS/MS data obtained for molecules adducted with two ammonium cations.