Developments in ion mobility spectrometry–mass spectrometry

Ion mobility spectrometry (IMS) has been used for over 30 years as a sensitive detector of organic compounds. The following is a brief review of IMS and its principles with an emphasis on its usage when coupled to mass spectrometry. Since its inception, IMS has been interfaced with quadrupole, time-of-flight, and Fourier-transform ion cyclotron resonance mass spectrometry. These hybrid instruments have been employed for the analysis of a variety of target analytes, including biomolecules, explosives, chemical warfare degradation products, and illicit drugs.     

Liquid chromatography–electrochemistry–mass spectrometry of polycyclic aromatic hydrocarbons

An efficient method for fast elucidation of the electrochemical reactions of polycyclic aromatic hydrocarbons (PAH) has been set up by applying post-column electrochemistry in liquid chromatography–mass spectrometry (LC–MS). With this set-up strong improvement of sensitivity in the LC–MS analysis of PAH is observed. Due to their low redox potentials, the non-polar PAH are converted into the respective radical cations, which may further react with constituents of the mobile phase and in additional electrochemical oxidation steps. Among other products, mono-, di-, and trioxygenated species are observed in aqueous solutions, alkoxylated compounds in alcohols, and solvent adducts in the presence of acetonitrile. While more different products are observed by using atmospheric pressure chemical ionization in the positive-ion mode (APCI(+)), the deprotonation of hydroxylated species results in very clear spectra in the negative-ion mode (APCI(–)). Deuterated PAH and deuterated solvents were used to gain additional information on the formation of the reaction products.     

Study of the microbiodegradation of terpenoid resin-based varnishes from easel painting using pyrolysis–gas chromatography–mass spectrometry and gas chromatography–mass spectrometry

The alterations produced by microbiological attack on terpenoid resin-based varnishes from panel and canvas paintings have been evaluated using pyrolysis–gas chromatography–mass spectrometry (Py–GC–MS) and gas chromatography–mass spectrometry (GC–MS). The proposed methods include the on-line derivatisation of drying oils and diterpenoid resins using hexamethyldisilazane during pyrolysis and the application of methyl chloroformate as a derivatisation reagent for triterpenoid resins in GC–MS. Two types of specimens, consisting of model oil medium prepared from linseed oil and model spirit varnishes prepared from colophony and mastic resins dissolved in turpentine, have been used as reference materials. For a series of specimens upon which different genera of bacteria and fungi were inoculated and encouraged to grow, analyses indicated that no mechanisms that commonly occur during the attack of enzymes on drying oils and terpenoid biodegraders were observed to occur in the oil medium and varnishes studied. Thus, the degradation pathways observed in the performed trials usually occur as consequence of natural ageing. Specific trials consisting of the application of biocides to uninoculated colophony varnish resulted in the identification of processes that produce undesirable degradation of the varnish due to interactions between the biocide and the varnish components. Finally, the studied biocides—Biotin, New-Des and Nipagine—generally exhibited good inhibiting effects on the microorganisms studied, although some interesting differences were found between them regarding the application method and type of biocide.     

Recent developments in protein–ligand affinity mass spectrometry

This review provides an overview of direct and indirect technologies to screen protein–ligand interactions with mass spectrometry. These technologies have as a key feature the selection or affinity purification of ligands in mixtures prior to detection. Specific fields of interest for these technologies are metabolic profiling of bioactive metabolites, natural extract screening, and the screening of libraries for bioactives, such as parallel synthesis libraries and small combichem libraries. The review addresses the principles of each of the methods discussed, with a focus on developments in recent years, and the applicability of the methods to lead generation and development in drug discovery.     

Neurosteroid analysis by gas chromatography–atmospheric pressure photoionization–tandem mass spectrometry

A new and simple APPI interface employing commercially available hardware is used to combine GC to MS. The feasibility of the method is demonstrated in the analysis of urine samples for neurosteroids as their trimethylsilyl (TMS) derivatives. The effect of different dopants (chlorobenzene, toluene, anisole) on the ionization of the TMS derivatives was investigated. With chlorobenzene, the TMS derivatives produced intense molecular ions with minimal fragmentation, and chlorobenzene was selected as best dopant. Protonated molecules in addition to intense molecular ions were produced with toluene and anisole. The performance of the method was verified in the analysis of human urine samples. Chromatographic performance was good with peak half-widths of 3.6–4.3 s, linearity (r² > 0.990) was acceptable, limits of detection (LODs) were in the range of 0.01–10 ng mL⁻¹, and repeatability was good with relative standard deviations (rsd%) below 22%. The results show that the method is well suited for the determination of neurosteroids in biological samples.     

Investigating direct current potentials that affect native protein conformation during trapped ion mobility spectrometry–mass spectrometry

Trapped ion mobility spectrometry–time-of-flight mass spectrometry (TIMS-TOFMS) has emerged as a tool to study protein conformational states. In TIMS, gas-phase ions are guided across the IM stages by applying direct current (DC) potentials (D1–6), which, however, might induce changes in protein structures through collisional activation. To define conditions for native protein analysis, we evaluated the influence of these DC potentials using the metalloenzyme bovine carbonic anhydrase (BCA) as primary test compound. The variation of DC potentials did not change BCA-ion charge and heme content but affected (relative) charge-state intensities and adduct retention. Constructed extracted-ion mobilograms and corresponding collisional cross-section (CCS) profiles gave useful insights in (alterations of) protein conformational state. For BCA, the D3 and D6 potential (which are applied between the deflection transfer and funnel 1 [F1] and the accumulation exit and the start of the ramp, respectively) had most profound effects, showing multimodal CCS distributions at higher potentials indicating gradual unfolding. The other DC potentials only marginally altered the CCS profiles of BCA. To allow for more general conclusions, five additional proteins of diverse molecular weight and conformational stability were analyzed, and for the main protein charge states, CCS profiles were constructed. Principal component analysis (PCA) of the obtained data showed that D1 and D3 exhibit the highest degree of correlation with the ratio of folded and unfolded protein (F/U) as extracted from the mobilograms obtained per set D potential. The correlation of D6 with F/U and protein charge were similar, and D2, D4, and D5 showed an inverse correlation with F/U but were correlated with protein charge. Although DC boundary values for induced conformational changes appeared protein dependent, a set of DC values could be determined, which assured native analysis of most proteins.     

Monitoring chlorophenols in industrial effluents by solid-phase microextraction–gas chromatography–mass spectrometry

A method for the determination of 19 chlorophenols in industrial effluents samples using solid-phase microextraction (SPME) coupled to gas chromatography–mass spectrometry has been developed. Four kinds of different SPME fibres have been studied. Among them, the polyacrylate and carbowax®-divinylbenzene fibres were the most adequate. The extraction process was optimized by means of the experimental design, which allows the study of a large number of factors with a reasonable number of experiments. The optimized method allows the determination of the studied chlorophenols in complex matrices with a high organic content with detection limits down to 0.07?ng?mL^?1 and RSD ranging from 4.4% to 13.8%. The recovery studies with spiked real effluent samples at low levels of chlorophenols ranged from 59.8% to 142.1% for the lowest level (0.5?ng?mL^?1) and from 79.6% to 115.8% for the highest spiked level (2?ng?mL^?1). These results show the suitability of the proposed method to monitor chlorophenols in complex samples. 2,4,5-TCP was detected at concentrations close to its limits of detection in effluents coming from an oil refinery.     

Identification of monoacylglycerol regio-isomers by gas chromatography–mass spectrometry

Monoacylglycerols (MAGs) are lipids found in trace amounts in plants and animal tissues. While they are widely used in various industrial applications, accurate determination of the regio-specific distribution is hindered by the lack of stable, commercially available standards. Indeed, unsaturated β-MAG (or Sn-2 MAG) readily undergoes isomerization into α-MAG (acyl chain is attached to the Sn-1 or the Sn-3 position). In the present study, we describe structural elucidation of α- and β-regio-isomers of monopalmitoyl-glycerol (MAG C16:0) as model compounds in their silylated forms using gas chromatography–mass spectrometry (GC–MS) with electronic impact (EI) ionization. MS fragmentation of α-MAG C16:0 is characterized by the loss of methylene(trimethylsilyl)oxonium (103 amu) and the consecutive loss of acyl chain yielding a fragment ion at m/z 205. The fragmentation pattern of β-MAG C16:0 shows a series of diagnostic fragments at m/z 218, 203, 191 and 103 that are not formed from the α-isomer and hereby enable reliable distinction of these regio-isomers. Possible fragmentation scenarios are postulated to explain the formation of these marker ions, which were also applied to characterize the regio-isomer composition of a complex mixture of MAG sample containing n-3 long-chain polyunsaturated fatty acids.     

Nanoscale reversed-phase liquid chromatography–mass spectrometry of permethylated N-glycans

Reversed-phase liquid chromatography on the nanoscale coupled to electrospray tandem mass spectrometry was used to analyse a mixture of four commercial glycan standards, and the method was further adapted to N-glycans enzymatically released from alpha-1-acid glycoprotein and immunoglobulin gamma. Glycans were permethylated to enable their separation by reversed-phase chromatography and to facilitate interpretation of fragmentation data. Prior to derivatization of glycans by permethylation, they were reduced to cancel anomerism because, although feasible, it was not desired to separate α- and β-anomers. The effect of supplementing chromatographic solvent with sodium hydroxide to guide adduct formation was investigated. Raising the temperature in which the separation was performed improved chromatographic resolution and affected retention times as expected. It was shown by using the tetrasaccharides sialyl Lewis X and sialyl Lewis A that reversed-phase chromatography could achieve the separation of methylated isobaric glycan analytes. Isobaric glycans were detected among the N-glycans of immunoglobulin gamma and further analysed by tandem mass spectrometry.     

Oligosaccharide analysis by graphitized carbon liquid chromatography–mass spectrometry

Structural analysis of complex mixtures of oligosaccharides using tandem mass spectrometry is regularly complicated by the presence of a multitude of structural isomers. Detailed structural analysis is, therefore, often achieved by combining oligosaccharide separation by HPLC with online electrospray ionization and mass spectrometric detection. A very popular and promising method for analysis of oligosaccharides, which is covered by this review, is graphitized carbon HPLC–ESI-MS. The oligosaccharides may be applied in native or reduced form, after labeling with a fluorescent tag, or in the permethylated form. Elution can be accomplished by aqueous organic solvent mixtures containing low concentrations of acids or volatile buffers; this enables online ESI-MS analysis in positive-ion or negative-ion mode. Importantly, graphitized carbon HPLC is often able to resolve many glycan isomers, which may then be analyzed individually by tandem mass spectrometry for structure elucidation. While graphitized carbon HPLC–MS for glycan analysis is still only applied by a limited number of groups, more users are expected to apply this method when databases which support structural assignment become available.

Gas chromatography–mass spectrometry analysis of pheomelanin degradation products

Melanoma is most rapidly increasing in the white population and people with pheomelanin skin type are at high risk to develop melanoma. However, little is known about the pheomelanin structure and function, and further elucidation of this melanin is therefore an important task. A GC/MS method was developed based on hydriodic acid hydrolysis of pheomelanin in the urine. Derivatization was performed with ethyl chloroformate and ethanol:pyridine (4:1, v/v). N,O-Ethoxycarbonyl-ethyl esters were extracted with chloroform and analyzed by GC/MS. 4-Amino-3-hydroxyphenylalanine and 3-amino-4-hydroxyphenylalanine together with one benzothiazinone and two benzothiazole compounds were detected and identified in hydrolyzed samples of synthetic pheomelanin and melanin from the urine of a patient with melanoma. These findings strongly suggest that heterocyclic pheomelanin-type units are incorporated in the pigment structures.     

Development of electrothermal atomic absorption spectrometry in 2005–2016

The review covers publications of 2005–2016 on achievements in the development of electrothermal atomic absorption spectrometry (ETAAS). The main directions in the authors’ opinion are revealed, i.e., (1) improvements of the method and equipment and (2) studies of thermochemical processes in a graphite furnace. In the first group, the authors consider high- and low-resolution continuum source atomic absorption spectrometry, diode laser atomic absorption spectrometry, new designs of electrothermal atomizers, and new devices for ETAAS. Studies of mechanisms of element atomization, formation of analytical signals, and action of chemical modifiers belong to the second group.     

Novel aspects of quantitation of immunogenic wheat gluten peptides by liquid chromatography–mass spectrometry/mass spectrometry

A novel, specific and sensitive non-immunological liquid chromatography–mass spectrometry (LC–MS) based assay has been developed to detect and quantify trace levels of wheat gluten in food and consumer products. Detection and quantification of dietary gluten is important, because gluten is a principle trigger of a variety of immune diseases including food allergies and intolerances. One such disease, celiac sprue, can cause intestinal inflammation and enteropathy in patients who are exposed to dietary gluten. At present, immunochemistry is the leading analytical method for gluten detection in food. Consequently, enzyme-linked immunosorbent assays (ELISAs), such as the sandwich or competitive type assays, are the only commercially available methods to ensure that food and consumer products are accurately labeled as gluten-free. The availability of a comprehensive, fast and economic alternative to the immunological ELISA may also facilitate research towards the development of new drugs, therapies and food processing technologies to aid patients with gluten intolerances and for gluten-free labeling and certification purposes. LC–MS is an effective and efficient analytical technique for the study of cereal grain proteins and to quantify trace levels of targeted dietary gluten peptides in complex matrices. Initial efforts in this area afforded the unambiguous identification and structural characterization of six unique physiologically relevant wheat gluten peptides. This paper describes the development and optimization of an LC–MS/MS method that attempts to provide the best possible accuracy and sensitivity for the quantitative detection of trace levels of these six peptides in various food and consumer products. The overall performance of this method was evaluated using native cereal grains. Experimental results demonstrated that this method is capable of detecting and quantifying select target peptides in food over a range from 10 pg/mg to 100 ng/mg (corresponding to approximately 0.01–100 ppm). Limits of detection (LOD) and quantification (LOQ) for the six target peptides were determined to range from 1 to 30 pg/mg and 10–100 pg/mg respectively. Reproducibility of the assay was demonstrated by evaluation of calibration data as well as data collected from the analysis of quality control standards over a period of four consecutive days. The average coefficient of determination (R²) for each peptide was consistently found to be >0.995 with residuals ranging from approximately 80% to 110%. Spike recovery data for each peptide in various matrices was evaluated at a concentration level near the approximate LOQ for each, as well as at higher concentration levels (30 and 60 ng/mg). The average range of accuracy of detection for all peptides at the lower concentration level was determined to be 90% (±11), while accuracy at the 30 and 60 ng/mg levels was 98% (±5%) and 98% (±3%), respectively. The usefulness and capabilities of this method are presented in a practical application to prospectively screen a variety of common commercially available (native and processed) gluten-containing and gluten-free foods and products.     

Non-enzymatic posttranslational modifications of bovine serum albumin by oxo-compounds investigated by high-performance liquid chromatography–mass spectrometry and capillary zone electrophoresis–mass spectrometry

Non-enzymatic posttranslational modifications of bovine serum albumin (BSA) by various oxo-compounds (glucose, ribose, glyoxal and glutardialdehyde) have been investigated using high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE). Both of these methods used mass spectrometric (MS) detection. Three enzymes (trypsin, pepsin, proteinase K) were used to digest glycated BSA. The extent of modification depended on the selected oxo-compound. Reactivity increased progressively from glucose to glutardialdehyde (glucose < ribose < glyoxal < glutardialdehyde). Carboxymethylation of lysine (CML) was the main type of modification detected. The HPLC/MS method achieved higher coverage and a larger amount of CML was identified compared to CZE/MS.     

Characterization of impurities in tobramycin by liquid chromatography–mass spectrometry

The characterization of unknown impurities present in tobramycin by liquid chromatography (LC) coupled with mass spectrometry (MS) is described. A reversed-phase (RP)-LC method using a volatile mobile phase containing a perfluorinated ion-pair reagent was developed and coupled with an ion trap mass spectrometer. The structures of the unknown impurities were deduced by comparison of their fragmentation patterns with those of the available reference substances obtained by LC–MSⁿ experiments.     

Quantitative determination of capsaicinoids by liquid chromatography–electrospray mass spectrometry

Eight naturally occurring capsaicinoids have been determined in Capsicum by use of high-purity standards, with norcapsaicin as an internal standard. The solid standards were rigorously checked for purity. The sensitivity of electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI), and coordination ion-spray (CIS; with silver) toward the capsaicinoids were measured and compared. The highest sensitivity was found for positive-ion ESI. Method validation of the liquid chromatography–ESI-mass spectrometry (LC–ESI-MS) determination is reported, including tests for repeatability (4%), detection limit (5 pg injected), linear range (20–6 ng injected), quantitation (excellent linearity; <2% relative standard deviation), and recovery (99–103%). The major and minor capsaicinoids in a commercial plant extract and in chili pepper fruits were quantified.Electronic Supplementary Material Supplementary material is available for this article at