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The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated., Author summary Today’s diagnosis of infection with the filarial parasite Onchocerca volvulus mainly depends on the microscopic analysis of skin biopsies and serological testing. The work presented here describes the use of multiple mass spectrometry-based screening methods (metabolomics and lipidomics) to search for biomarkers indicative of infection with Onchocerca volvulus. This resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine as biomarker for O. volvulus. Further evaluation of these biomarkers in a geographically distinct non-endemic population however invalidated the use of urine cis-cinnamoylglycine. These findings are of utmost importance as it not only opens new avenues in the development of non-invasive diagnostic tools for filarial infections, but also emphasizes the need for evaluation and validation of newly discovered biomarkers in different populations from different geographies.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550. ispartof: JACS AU vol:1 issue:6 pages:750-765 ispartof: location:United States status: published

Two low-pressure columns (Bio-Beads SX-3) and three high-pressure GPC columns were compared for clean-up of a wide range of pesticides in fatty matrices of vegetable or animal origin. The GPC fractions were analyzed by GC-MS/MS and LC-MS/MS without additional clean-up. The performance of the GPC clean-up on the five column types was compared in terms of solvent consumption, lipid removal, pesticide recovery and repeatability. It was found that for fatty matrices, mainly consisting of high molecular weight triglycerides i.e. most vegetable oils and animal fats, good fractionation is obtained for the majority of the pesticides. On the other hand, for fats and oils containing relatively high amounts of low molecular weight triglycerides, i.e. butter fat and palm kernel oil, none of the columns provided sufficient clean-up and cause interferences and system contamination, especially in the case of GC-MS/MS analysis. For the latter case, best results in terms of lipid removal and pesticide recovery were obtained on a set (2×300mmlength) of narrow bore (7.5mm ID) columns packed with 5µm PL Gel material. Column loadability is, however, much lower on that set of columns compared the other evaluated GPC columns, impairing overall method sensitivity.

Analytical pyrolysis combined with gas chromatography–mass spectrometry (Py-GC–MS) is an effective technique for studying combustion processes such as cigarette smoking. Direct pyrolysis of tobacco leaf results in a very complex mixture of organic compounds containing volatile, semi-volatile and non-volatile compounds. Consequently, detecting and identifying the main tobacco aroma compounds and their formation pathways are very difficult. An innovative workflow that can elucidate the aroma compounds formed during the combustion of tobacco is presented. The workflow consists of LC fractionation of a tobacco leaf extract, recombination of fractions in a fraction omission approach and Py-GC–MS. Using multivariate statistical data analysis, the fractions that contribute most to the formation of typical cigarette smoke aromas could be identified. In addition, GC–MS analysis of derivatized LC fractions could be used to identify the precursors of these aroma compounds. The analytical approach is suitable to correlate the effect of different classes of compounds in tobacco to the typical aroma compounds in cigarette smoke.

Gas chromatography coupled to isotope ratio mass spectrometry after on-line combustion (GC-C-IRMS) and high temperature conversion (GC-HTC-IRMS) is used for compound specific isotope ratio determination. This determination can only be performed successfully if the target solutes are fully resolved from other compounds. A new instrumental set-up consisting of heart-cutting two-dimensional GC based on capillary flow technology and a low thermal mass GC oven in combination with an isotope ratio mass spectrometer is presented. Capillary flow technology was also used in all column and interface connections for robust and leak-free operation. The new configuration was applied to the characterization of wax compounds in tobacco leaf and corresponding smoke samples. It is demonstrated that high accuracy is obtained, both in the determination of δ(13)C and δ(2)H values, allowing the study of biosynthesis and delivery mechanisms of naturally occurring compounds in tobacco.

An LC-MS based method for the profiling and characterization of ceramide species in the upper layer of human skin is described. Ceramide samples, collected by tape stripping of human skin, were analyzed by reversed-phase liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry operated in both positive and negative electrospray ionization mode. All known classes of ceramides could be measured in a repeatable manner. Furthermore, the data set showed several undiscovered ceramides, including a class with four hydroxyl functionalities in its sphingoid base. High-resolution MS/MS fragmentation spectra revealed that each identified ceramide species is composed of several skeletal isomers due to variation in carbon length of the respective sphingoid bases and fatty acyl building blocks. The resulting variety in skeletal isomers has not been previously demonstrated. It is estimated that over 1000 unique ceramide structures could be elucidated in human stratum corneum. Ceramide species with an even and odd number of carbon atoms in both chains were detected in all ceramide classes. Acid hydrolysis of the ceramides, followed by LC-MS analysis of the end-products, confirmed the observed distribution of both sphingoid bases and fatty acyl groups in skin ceramides. The study resulted in an accurate mass retention time library for targeted profiling of skin ceramides. It is furthermore demonstrated that targeted data processing results in an improved repeatability versus untargeted data processing (72.92% versus 62.12% of species display an RSD < 15%).

A separation method based on ion-pairing liquid chromatography was combined with both elemental (inductively coupled plasma mass spectrometry (ICP-MS)) and molecular (electrospray tandem mass spectrometry (ES-MS-MS)) mass spectrometry in order to unravel the identity of the Se-species present in the complex matrix of Brazil nuts rich in Se. Via enzymatic digestion, Se-species were released from the matrix. Subsequently the species were separated and the Se was monitored on-line by ICP-MS. By HPLC–ES-MS-MS, the species were identified based on their molecular mass and their specific product ions. The main compound was identified as Se-Methionine. Another compound was identified as Se-Cystine, partly on the basis of the isotopic pattern of Se. This research was further extended to the analyses of in vitro gastrointestinal digests of the Brazil nuts. These digests were analyzed for their Se-content and screened for the presence of the different Se-species by HPLC–ICP-MS. In both the gastric and the intestinal digests, we were able to identify the Se-species as Se-Methionine and Se-Cystine by HPLC–ES-MS-MS. By coupling HPLC to both elemental and molecular mass spectrometry, the species present in Brazil nuts and supposedly extractable by our body were fully characterized.

The metabolism of selenium (Se) in the human body has yet not completely been unravelled and hence, an efficient method for characterization and on-line monitoring of the main Se-compound in human urine after consumption of Se-rich food was developed. Total Se-concentration in human urine after consumption of several Se-rich products was measured with inductively coupled plasma mass spectrometry (ICP-MS). The highest Se concentration in urine was observed after 4–10 h. The urine samples were brought onto a reversed phase column and the Se was detected by ICP-MS. Parameters for liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) measurements were optimized by using commercially available sugars, because it is known that some of the urinary metabolites contain a sugar moiety. In order to characterize the predominant Se-metabolite, it was necessary to extensively clean-up the sample and preconcentrate the species. The main metabolite was measured on its precursor ion on three different m / z according to three isotopes of Se. Relative peak surfaces matched the relative abundances of the isotopes. The product ions could be measured in a human urine sample in accordance to the product ions of the commercially available sugars. Moreover, the evidence of a selenosugar was demonstrated by the use of the Se-isotopes when measuring the product ions. LC-ESI-MS-MS was proven to be very efficient for the characterization of the main urinary Se-metabolite and can be used for on-line monitoring of the compound in urine samples. The method can be extended for clinical screening after consumption of Se-(en)rich(ed) food by use of the Se-isotopic profile and/or of the typical product ions of (methyl)- N -acetyl-hexosamines.

Liquid chromatography coupled to a hydride generation atomic fluorescence spectrometer has been applied for the speciation of Se in extracts of Saccharomyces cerevisiae. In order to develop a method which allows the separation of the compounds and detection of the element, seven Se standards were used: Se-methionine (Se-Met), Se-cystine (Se-(Cys)2), Se-cystamine (Se-Cya), Se-methylselenocysteine (Se-MeSeCys), Se-ethionine (Se-Et), selenate (SeVI), selenite (SeIV). Optimal chromatographic results were obtained with reversed-phase chromatography on an XTerra C18 column using a positively charged ion-pairing agent. It was observed that for these standards precise control of the pH was of utmost importance. Attention was devoted to the compatibility of the mobile phase with hydride generation. Efficient formation of the hydrides was obtained by optimisation of different parameters. The redox mixture which allowed optimum conversion of all different species was HBr–KBrO3. To assist in the conversion of the compounds, on-line microwave digestion was applied. The detection limits obtained for the standards were: 0.8 µg Se l−1 for selenite(IV); 1.3 µg Se l−1 for selenate(VI); 1.2 µg Se l−1 for Se-methionine; 1.2 µg Se l−1 for Se-cystine; 1.3 µg Se l−1 for Se-cystamine; and 1.1 µg Se l−1 for Se-methylselenocysteine, respectively. Se-compounds in Saccharomyces cerevisiae were extracted by hot water (50 °C) or proteolytic digestion with protease XIV (37 °C). The method developed for separation and elemental detection was applied to these extracts in order to distinguish between the different species extracted from the yeast matrix. Total Se concentration in the extracts was measured with pneumatic nebulization-inductively coupled plasma-mass spectrometry (PN-ICP-MS). Species transformation was investigated by analysing extracts preserved at 2 different temperatures (−20 °C and 4 °C). Only those extracts kept at −20 °C proved to be unchanged.

Slab-gel electrophoresis has been applied to the speciation of vanadium in serum. The electrophoresis separation is an adaptation of the blue native polyacrylamide gel electrophoresis separation necessary to ensure the stability of the vanadium-protein complex; Coomassie blue was used to shift the charges of the proteins and to stabilize the vanadium complex. The detection of the vanadium species was made possible by the use of the (48)V radiotracer and the phosphor-screen technology. The method was first developed using transferrin, incubated with (48)V, as a model. After it was proved that the vanadium-transferrin complex was stable during separation, the method was validated by separating serum incubated with (48)V. The efficiency of the separation was assessed according to two parameters: resolution and conservation of the species. First, the resolution of the separation was as expected from a native separation. Second, the release of free vanadium from the transferrin complex, which was the main vanadium species expected, was negligible, which proves that the species remain intact during separation. In accordance with the literature, it was found that vanadium binds to transferrin in incubated serum at these low concentrations.

Two-dimensional high-resolution gel electrophoresis (2DE) has been applied to the fractionation of 75Se-containing proteins in yeast, grown in 75Se-containing medium, and autoradiography was used for detection of the 75Se-containing proteins. Gel filtration and ultrafiltration were used to check whether the selenium side-chains were stable in the presence of the chemicals used for lysis and 2DE. The mass distribution of the selenium-containing proteins was estimated by use of gel filtration and the results were compared with the distribution obtained by 2DE. A 2DE map of selenium-containing proteins in yeast is presented, and compared with a total protein map of yeast.

Three selenium species (selenomethionine, selenocystine and selenocystamine) were studied for their behaviour in the presence of the reducing agents dithiothreitol and tributylphosphine, and of a derivatising agent, iodoacetic acid. These chemicals are commonly used in proteome analysis by means of gel electrophoresis. To study the effect of these chemicals on the selenospecies, two separation techniques were used, capillary electrophoresis and liquid chromatography, both with ICP-MS as an element-specific detector. Electrospray-mass spectrometry provided molecular information. Tributylphosphine (TBT) was shown to partly reduce selenocystine to selenocysteine, and iodoacetic acid to derivatise all the species, with conservation of the species information. Dithiothreitol (DTT), on the contrary, partly sequestered selenium from the species to form a DTT–Se molecule.

Personal care products (PCPs), such as soaps, perfumes, cosmetics, lotions, etc., contain a variety of chemicals that have been described as potentially hormone disrupting chemicals. Therefore, it is important to assess the internal exposure of these chemicals in humans. Within the 2nd Flemish Environment and Health Study (FLEHS II, 2007–2011), the human exposure to three classes of pollutants that are present in a wide variety of PCPs – i.e. polycyclic musks (galaxolide, HHCB and tonalide, AHTN in blood), parabens (urinary para -hydroxybenzoic acid, HBA) and triclosan (urinary TCS) – was assessed in 210 Flemish adolescents (14–15 years) and in 204 adults (20–40 years) randomly selected from the general population according to a stratified two stage clustered study design. The aim of this study was to define average levels of exposure in the general Flemish population and to identify determinants of exposure. Average levels (GM (95% CI)) in the Flemish adolescents were 0.717 (0.682–0.753) μg/L for blood HHCB; 0.118 (0.108–0.128) μg/L for blood AHTN; 1022 (723–1436) μg/L for urinary HBA and 2.19 (1.64–2.92) μg/L for urinary TCS. In the adults, levels of HBA were on average 634 (471–970) μg/L. Inter-individual variability was small for HHCB and AHTN, intermediate for HBA, and large for TCS. All biomarkers were positively associated with the use of PCPs. Additionally, levels of HHCB and AHTN increased with higher educational level of the adolescents. Both in adults and adolescents, urinary HBA levels were negatively correlated with BMI. We define here Flemish exposure values for biomarkers of PCPs, which can serve as baseline exposure levels to identify exposure trends in future biomonitoring campaigns.

Arylamines and aminopyridines form a class of potentially genotoxic impurities (PGIs) that can be present at trace levels in active pharmaceutical ingredients (APIs). A generic method was developed that allows the analysis of a selected set of these solutes at sub-ppm level relative to the drug substance. A highly concentrated solution of the pharmaceutical compound is analyzed by LC-MS using a single quadrupole mass spectrometer in the selected ion monitoring (SIM) mode. Since a number of target compounds show little or no retention in the reversed-phase LC setup, a fast and simple derivatization procedure using hexylchloroformate was applied. The amide derivatives of the PGI result in a higher molecular weight (more specific ion for SIM) and better chromatographic behavior. The methodology, consisting of a dual run on respectively a non-derivatized and a derivatized sample, was validated and applied to a selection of pharmaceutical substances. The method was found to be sufficiently sensitive and robust and is applicable in a QA/QC environment.

A method based on solid phase extraction (SPE) followed by liquid chromatography–electrospray ionisation tandem mass spectrometry for the determination of the nonapeptides arginine vasotocin (AVT) and isotocin (IT) in brains of three-spined sticklebacks ( Gasterosteus aculeatus ) is described. Separation and detection were optimized using synthetic standards. Limits of detection (LOD) for standard solutions were 160 pg mL −1 for AVT and 250 pg mL −1 for IT. The SPE procedure hardly affected the LODs for standard solutions. Mainly because of ion suppression, LODs for AVT and IT in brains were approximately 5 and 25 pg mg −1 , respectively. The concentrations determined in the brain of several fishes ranged from 10 to 500 pg mg −1 for AVT and from 400 to 4000 pg mg −1 for IT.

Especially in the last decade, a vast number of papers on Se and its role in health issues have been published. This review gives a brief, critical overview of the main analytical findings reported in these papers. Of particular interest is the Se content in different food sources worldwide and the extent to which their consumption is reflected in the Se content of human tissues and body fluids. Several food sources, both natural (Brazil nuts, garlic, Brassica juncea) and Se-enriched (yeast-based supplements), are discussed as to origin, characteristics, Se metabolism and impact of their consumption on the human body. The continuous development of new and improvement of existing analytical techniques has provided different powerful tools to unravel the Se species and their function. An up-to-date literature study on Se speciation analysis is given, illustrating how analytical chemistry in its different facets aids in the identification of Se compounds and provides insight into the complete metabolic pathway of Se throughout the human body. This review includes a detailed image of the current state-of-the-art of Se speciation analysis in these food sources and in human tissues and body fluids.

Liquid chromatography (LC) hyphenated with both elemental and molecular mass spectrometry has been used for Se speciation in Se-enriched garlic. Different species were separated by ion-pair liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS) after hot-water extraction. They were identified by on-line reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (RPLC-ESI-MS-MS). Se-methionine and Se-methylselenocysteine were determined by monitoring their product ions. Another compound, gamma-glutamyl-Se-methylselenocysteine, shown to be the most abundant form of Se in the garlic, was determined without any additional sample pre-treatment after extraction and without the need for a synthesized standard. Product ions for this dipeptide were detected by LC-ESI-MS-MS for three isotopes of Se-78 Se, 80Se: and 82Se. The method was extended to the species extracted during in-vitro gastrointestinal digestion. Because both Se-methylselenocysteine and gamma-glutamyl-Se-methylselenocysteine have anticarcinogenic properties, their extractability and stability during human digestion are very important. Garlic was also treated with saliva, to enable detection and analysis of species extracted during mastication. Detailed information on the extractability of selenium species by both simulated gastric and intestinal fluid are given, and variation of the distribution of Se among the different species with time is discussed. Although the main species in garlic is the dipeptide gamma-glutamyl-Se-methylselenocysteine, Se-methylselenocysteine is the main compound present in the extracts after treatment with gastrointestinal fluids. Two more, so far unknown compounds were observed in the chromatogram. The extracted species and their transformations were analysed by combining LC-ICP-MS and LC-ESI-MS-MS. In both the simulated gastric and intestinal digests, Se-methionine, Se-methylselenocysteine, and gamma-glutamyl-Se-methylselenocysteine could be determined by LC-ESI-MS-MS by measuring their typical product ions.

On-line monitoring of six Se-compounds was accomplished by using an XTerra MS C18 column coupled to electrospray tandem mass spectrometry (ES-MS-MS). In view of the nature of the compounds, the positively charged ion pairing agent tetraethylammoniumchloride (TEACl) was added to the mobile phase. The HPLC-ES-MS-MS method was optimized with six commercially available Se-compounds. Substitution of the analytical column by the narrowbore type significantly enhanced the sensitivity of the method. We were able to detect the m/z of these six molecules on-line. Furthermore, all product ions could be monitored. The method was applied to three different yeast-based supplements. They were submitted to proteolytic digestion and screened for their Se-content by HPLC-HG-AFS (hydride generation-atomic fluorescence spectrometry). By application of on-line narrowbore HPLC-electrospray tandem mass spectrometry, the main compound present in these three supplements, Se-Methionine, could be measured on its m/z and its product ions. The method can be further extended for on-line measurement of different Se-species in complex matrices

A method was developed allowing the separation, detection and identification of Se species extracted from yeast supplements during simulated digestion processes. The in vitro gastric and intestinal digests were studied for their Se compounds by successive high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS) and high-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES-MS-MS) analyses. The conditions for the separation were chosen as to be compatible with both ICP-MS and ES-MS-MS detection. HPLC-ICP-MS was used to screen the extracts for their Se content. By means of HPLC-ES-MS-MS, the compounds extracted were identified on-line according to their retention time, m/ z of the molecular ion and the presence of typical product ions. From these results, it was clear that the main compound extracted by both gastric and intestinal fluid was Se-methionine, which was also the main Se compound extracted by proteolytic digestion from the yeast supplements. Two other minor compounds could be identified as Se-cystine and Se(O)-methionine, a degradation product of Se-methionine.

Slab-gel electrophoresis has been applied to the speciation of vanadium in serum. The electrophoresis separation is an adaptation of the blue native polyacrylamide gel electrophoresis separation necessary to ensure the stability of the vanadium-protein complex; Coomassie blue was used to shift the charges of the proteins and to stabilize the vanadium complex. The detection of the vanadium species was made possible by the use of the (48)V radiotracer and the phosphor-screen technology. The method was first developed using transferrin, incubated with (48)V, as a model. After it was proved that the vanadium-transferrin complex was stable during separation, the method was validated by separating serum incubated with (48)V. The efficiency of the separation was assessed according to two parameters: resolution and conservation of the species. First, the resolution of the separation was as expected from a native separation. Second, the release of free vanadium from the transferrin complex, which was the main vanadium species expected, was negligible, which proves that the species remain intact during separation. In accordance with the literature, it was found that vanadium binds to transferrin in incubated serum at these low concentrations.