Ionization High-resolution Tandem Mass Spectrometry Research Articles

The blistering warfare agent O-mustard (agent T) generates protein-adducts with human serum albumin useful for biomedical verification of exposure and forms intramolecular cross-links.

The highly blistering sulfur mustard analogue agent T (bis(2-chloroethylthioethyl) ether), also known as O-mustard or oxy-mustard, is a common impurity in military grade sulfur mustard (SM) and a component of mixtures such as "HT" that are still found in old munitions. Together with sesquimustard (Q), it is the most important SM analogue and tightly regulated as a Schedule 1 chemical under the Chemical Weapons Convention. We report the adducts of T with nucleophilic Cys34 and other residues in human serum albumin (HSA) formed in vitro. A micro liquid chromatography electrospray ionization high-resolution tandem-mass spectrometry method (µLC-ESI MS/HRMS) was developed for the detection and identification of biomarker peptides alkylated by a T-derived hydroxyethylthioethyloxyethylthioethyl (HETEOETE)-moiety (as indicated by an asterisk below). Following proteolysis of T-exposed human plasma with pronase, the dipeptide Cys34*Pro and the single amino acid residue His* were produced. The use of proteinaseK yielded Cys34*ProPhe and theuse of pepsin generated ValThrGlu48*Phe, AlaGlu230*ValSerLysLeu, and LeuGlyMet329*Phe. Corresponding peptide-adducts of SM and Q were detected in a common workflow that in principle allowed the estimation of the mustard or mustard composition encountered during exposure. Novel adducts of Q at the Glu230 and Met239 residues were detected and are reported accordingly. Based on molecular dynamics simulations, we identified regular interactions of the Cys34(-HETEOETE)-moiety with several glutamic acid residues in HSA including Glu86, which is not an obvious interaction partner by visual inspection of the HSA crystal structure. The existence of this and other intramolecular cross-links was experimentally proven for the first time.

Evidence of nerve agent VX exposure in rat plasma by detection of albumin-adducts in vitro and in vivo

VX is a highly toxic organophosphorus nerve agent that reacts with a variety of endogenous proteins such as serum albumin under formation of adducts that can be targeted by analytical methods for biomedical verification of exposure. Albumin is phosphonylated by the ethyl methylphosphonic acid moiety (EMP) of VX at various tyrosine residues. Additionally, the released leaving group of VX, 2-(diisopropylamino)ethanethiol (DPAET), may react with cysteine residues in diverse proteins. We developed and validated a microbore liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (µLC-ESI MS/HR MS) method enabling simultaneous detection of three albumin-derived biomarkers for the analysis of rat plasma. After pronase-catalyzed cleavage of rat plasma proteins single phosphonylated tyrosine residues (Tyr-EMP), the Cys34(-DPAET)Pro dipeptide as well as the rat-specific LeuProCys448(-DPAET) tripeptide were obtained. The time-dependent adduct formation in rat plasma was investigated in vitro and biomarker formation during proteolysis was optimized. Biomarkers were shown to be stable for a minimum of four freeze-and-thaw cycles and for at least 24 h in the autosampler at 15 °C thus making the adducts highly suited for bioanalysis. Cys34(-DPAET)Pro was superior compared to the other serum biomarkers considering the limit of identification and stability in plasma at 37 °C. For the first time, Cys34(-DPAET)Pro was detected in in vivo specimens showing a time-dependent concentration increase after subcutaneous exposure of rats underlining the benefit of the dipeptide disulfide biomarker for sensitive analysis.

Determination of Phosphodiesterase Type-5 Inhibitors (PDE-5) in Dietary Supplements.

This study proposed a high-performance thin-layer chromatography (HPTLC) screening method to detect phosphodiesterase 5 (PDE-5) inhibitors as possible adulterant agents in various dietary supplements. Chromatographic analysis was performed on silica gel 60F254 plates using a mixture of ethyl acetate:toluene:methanol:ammonia in a volume ratio of 50:30:20:0.5 as a mobile phase. The system provided compact spots and symmetrical peaks of sildenafil and tadalafil with retardation factor values of 0.55 and 0.90, respectively. The analysis of products purchased from the internet or specialized stores demonstrated the presence of sildenafil, tadalafil, or both compounds in 73.3% of products, highlighting inadequacies and inconsistencies in the labeling, as all dietary supplements were declared to be natural. The results were confirmed using ultra-high-performance liquid chromatography coupled with a positive electrospray ionization high-resolution tandem mass spectrometry (UHPLC-HRMS-MS) method. Furthermore, in some samples, vardenafil and various analogs of PDE-5 inhibitors were detected using a non-target HRMS-MS approach. The results of the quantitative analysis revealed similar findings between the two methods, with adulterant quantities found to be similar to or higher than those in approved medicinal products. This study demonstrated that the HPTLC method is a suitable and economical method for screening PDE-5 inhibitors as adulterants in dietary supplements intended for sexual activity enhancement.

Assessing the Effectiveness of Chemical Marker Extraction from Amazonian Plant Cupuassu (Theobroma grandiflorum) by PSI-HRMS/MS and LC-HRMS/MS.

Employing a combination of liquid chromatography electrospray ionization and paper spray ionization high-resolution tandem mass spectrometry, extracts from cupuassu (Theobroma grandiflorum) pulp prepared with either water, methanol, acetonitrile or combinations thereof were subjected to metabolite fingerprinting. Among the tested extractors, 100% methanol extracted preferentially phenols and cinnamic acids derivatives, whereas acetonitrile and acetonitrile/methanol were more effective in extracting terpenoids and flavonoids, respectively. And while liquid chromatography- mass spectrometry detected twice as many metabolites as paper spray ionization tandem mass spectrometry, the latter proved its potential as a screening technique. Comprehensive structural annotation showed a high production of terpenes, mainly oleanane triterpene derivatives. of the mass spectra Further, five major metabolites with known antioxidant activity, namely catechin, citric acid, epigallocatechin-3'-glucuronide, 5,7,8-trihydroxyflavanone, and asiatic acid, were subjected to molecular docking analysis using the antioxidative enzyme peroxiredoxin 5 (PRDX5) as a model receptor. Based on its excellent docking score, a pharmacophore model of 5,7,8-trihydroxyflavanone was generated, which may help the design of new antioxidants.

Phosphonylated tyrosine and lysine residues as biomarkers of local exposure of human hair to the organophosphorus nerve agents sarin and VX.

We herein present for the first time a micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS) procedure to detect phosphonylated tyrosine (Tyr) and lysine (Lys) residues obtained from human hair exposed to organophosphorus nerve agents (OPNA). In general, toxic OPNA react with endogenous blood proteins causing the formation of adducts representing well-known targets for biomedical analysis to prove exposure. In contrast, no protein-derived biomarker has been introduced so far to document local exposure of hair. Accordingly, we developed and characterized a μLC-ESI MS/HR MS method for the analysis of scalp hair exposed to OPNA in vitro. Type I and Type II keratin from hair was dissolved during lysis, precipitated and subjected to pronase-catalyzed hydrolysis yielding single adducted Lys and in a much higher amount Tyr residues. Exposure to sarin caused the adduction of an isopropyl methylphosphonic acid moiety and exposure to VX yielded adducts of ethyl methylphosphonic acid, well suited as biomarkers of exposure. These were of appropriate stability in the autosampler for 24 h. The biomarker yield obtained from hair of six individuals as well as from hair of six different parts of the body of one individual (armpit, beard, leg, arm, scalp, and pubic) differed reasonably indicating the variable individual protein composition and structure of hair. Exposed hair stored at ambient temperature for 9weeks with contact to air and daylight showed stability of all adducts and therefore their suitability for verification of exposure.

Quantitation by Liquid Chromatography-Nanoelectrospray Ionization-High-Resolution Tandem Mass Spectrometry of Multiple DNA Adducts Related to Cigarette Smoking in Oral Cells in the Shanghai Cohort Study.

We developed a liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry (LC-NSI-HRMS/MS) method for simultaneous quantitative analysis of 5 oral cell DNA adducts associated with cigarette smoking: (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)-one (γ-OH-Acr-dGuo, 1) from acrolein; (6S,8S and 6R,8R)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxy-6-methylpyrimido[1,2-a]purine-10(3H)-one [(6S,8S)γ-OH-Cro-dGuo, 2; and (6R,8R)γ-OH-Cro-dGuo, 3] from crotonaldehyde; 1,N6-etheno-dAdo (4) from acrylonitrile, vinyl chloride, lipid peroxidation, and inflammation; and 8-oxo-dGuo (5) from oxidative damage. Oral cell DNA was isolated in the presence of glutathione to prevent artifact formation. Clear LC-NSI-HRMS/MS chromatograms were obtained allowing quantitation of each adduct using the appropriately labeled internal standards. The accuracy and precision of the method were validated, and the assay limit of quantitation was 5 fmol/μmol dGuo for adducts 1-4 and 20 fmol/μmol for adduct 5. The assay was applied to 80 buccal cell samples selected from those collected in the Shanghai Cohort Study: 40 from current smokers and 40 from never smokers. Significant differences were found in all adduct levels between smokers and nonsmokers. Levels of 8-oxo-dGuo (5) were at least 3000 times greater than those of the other adducts in both smokers and nonsmokers, and the difference between amounts of this adduct in smokers versus nonsmokers, while significant (P = 0.013), was not as great as the differences of the other DNA adducts between smokers and nonsmokers (P-values all less than 0.001). No significant relationship of adduct levels to risk of lung cancer incidence was found. This study provides a new LC-NSI-HRMS/MS methodology for the quantitation of diverse DNA adducts resulting from exposure to the α,β-unsaturated aldehydes acrolein and crotonaldehyde, inflammation, and oxidative damage which are all associated with carcinogenesis. We anticipate application of this assay in ongoing studies of the molecular epidemiology of cancers of the lung and oral cavity related to cigarette smoking.

Isolation of human TRPA1 channel from transfected HEK293 cells and identification of alkylation sites after sulfur mustard exposure

Transient receptor potential (TRP) channels are important in the sensing of pain and other stimuli. They may be triggered by electrophilic agonists after covalent modification of certain cysteine residues. Sulfur mustard (SM) is a banned chemical warfare agent and its reactivity is also based on an electrophilic intermediate. The activation of human TRP ankyrin 1 (hTRPA1) channels by SM has already been documented, however, the mechanism of action is not known in detail. The aim of this work was to purify hTRPA1 channel from overexpressing HEK293 cells for identification of SM-induced alkylation sites. To confirm hTRPA1 isolation, Western blot analysis was performed showing a characteristic double band at 125 kDa. Immunomagnetic separation was carried out using either an anti-His-tag or an anti-hTRPA1 antibody to isolate hTRPA1 from lysates of transfected HEK293 cells. The identity of the channel was confirmed by micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry. Following SM exposure, hTRPA1 channel modifications were found at Cys462 and Cys665, as well as at Asp339 and Glu341 described herein for the first time. Since Cys665 is a well-known target of hTRPA1 agonists and is involved in hTRPA1 activation, SM-induced modifications of cysteine, as well as aspartic acid and glutamic acid residues may play a role in hTRPA1 activation. Considering hTRPA1 as a target of other SM-related chemical warfare agents, analogous adducts may be predicted and identified applying the analytical approach described herein.

Quantitation of DNA Adducts Resulting from Acrolein Exposure and Lipid Peroxidation in Oral Cells of Cigarette Smokers from Three Racial/Ethnic Groups with Differing Risks for Lung Cancer.

The Multiethnic Cohort Study has demonstrated that the risk for lung cancer in cigarette smokers among three ethnic groups is highest in Native Hawaiians, intermediate in Whites, and lowest in Japanese Americans. We hypothesized that differences in levels of DNA adducts in oral cells of cigarette smokers would be related to these differing risks of lung cancer. Therefore, we used liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry to quantify the acrolein-DNA adduct (8R/S)-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3H)-one (γ-OH-Acr-dGuo, 1) and the lipid peroxidation-related DNA adduct 1,N6-etheno-dAdo (εdAdo, 2) in DNA obtained by oral rinse from 101 Native Hawaiians, 101 Whites, and 79 Japanese Americans. Levels of urinary biomarkers of nicotine, acrolein, acrylonitrile, and a mixture of crotonaldehyde, methyl vinyl ketone, and methacrolein were also quantified. Whites had significantly higher levels of γ-OH-Acr-dGuo than Japanese Americans and Native Hawaiians after adjusting for age and sex. There was no significant difference in levels of this DNA adduct between Japanese Americans and Native Hawaiians, which is not consistent with the high lung cancer risk of Native Hawaiians. Levels of εdAdo were modestly higher in Whites and Native Hawaiians than in Japanese Americans. The lower level of DNA adducts in the oral cells of Japanese American cigarette smokers than Whites is consistent with their lower risk for lung cancer. The higher levels of εdAdo, but not γ-OH-Acr-dGuo, in Native Hawaiian versus Japanese American cigarette smokers suggest that lipid peroxidation and related processes may be involved in their high risk for lung cancer, but further studies are required.

Increased acrolein-DNA adducts in buccal brushings of e-cigarette users.

DNA adducts are central in the mechanism of carcinogenesis by genotoxic agents. We compared levels of a DNA adduct of acrolein, a genotoxic carcinogen found in e-cigarette vapor, in oral cell DNA of e-cigarette users and non-users of any tobacco or nicotine product. e-Cigarette users and non-users visited our clinic once monthly for 6 months, and oral brushings and urine samples were collected. For this study, we analyzed oral cell DNA adducts from three monthly visits in e-cigarette users and non-users as confirmed by urinary cyanoethyl mercapturic acid and total nicotine equivalents. DNA was isolated from the oral brushings and analyzed by a validated liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method for the acrolein DNA adduct 8R/S-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10-(3H)-one (γ-OH-Acr-dGuo). The median value of this DNA adduct in the e-cigarette users was 179 fmol/µmol dGuo (range 5.0 - 793 fmol/µmol dGuo) while that for non-users was 21.0 fmol/µmol dGuo (range 5.0 - 539 fmol/µmol dGuo), P = 0.001. These results demonstrate for the first time that e-cigarette users have elevated levels of a carcinogen-DNA adduct in their oral cells.

Transthyretin as a target of alkylation and a potential biomarker for sulfur mustard poisoning: Electrophoretic and mass spectrometric identification and characterization.

For the verification of exposure to the banned blister agent sulfur mustard (SM) and the better understanding of its pathophysiology, protein adducts formed with endogenous proteins represent an important field of toxicological research. SM and its analogue 2-chloroethyl ethyl sulfide (CEES) are well known to alkylate nucleophilic amino acid side chains, for example, free-thiol groups of cysteine residues. The specific two-dimensional thiol difference gel electrophoresis (2D-thiol-DIGE) technique making use of maleimide dyes allows the staining of free cysteine residues in proteins. As a consequence of alkylation by, for example, SM or CEES, this staining intensity is reduced. 2D-thiol-DIGE analysis of human plasma incubated with CEES and subsequent matrix-assisted laser desorption/ionization time-of-flight (tandem) mass-spectrometry, MALDI-TOF MS(/MS), revealed transthyretin (TTR) as a target of alkylating agents. TTR was extracted from SM-treated plasma by immunomagnetic separation (IMS) and analyzed after tryptic cleavage by microbore liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS). It was found that the Cys10 -residue of TTR present in the hexapeptide C(-HETE)PLMVK was alkylated by the hydroxyethylthioethyl (HETE)-moiety, which is characteristic for SM exposure. It was shown that alkylated TTR is stable in plasma in vitro at 37°C for at least 14 days. In addition, C(-HETE)PLMVK can be selectively detected, is stable in the autosampler over 24 h, and shows linearity in a broad concentration range from 15.63 μM to 2mM SM in plasma in vitro. Accordingly, TTR might represent a complementary protein marker molecule for the verification of SM exposure.

Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro

Creatine kinase (CK) catalyzes the formation of phosphocreatine from adenosine triphosphate (ATP) and creatine. The highly reactive free cysteine residue in the active site of the enzyme (Cys283) is considered essential for the enzymatic activity. In previous studies we demonstrated that Cys283 is targeted by the alkylating chemical warfare agent sulfur mustard (SM) yielding a thioether with a hydroxyethylthioethyl (HETE)-moiety. In the present study, the effect of SM on rabbit muscle CK (rmCK) activity was investigated with special focus on the alkylation of Cys283 and of reactive methionine (Met) residues. For investigation of SM-alkylated amino acids in rmCK, micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry measurements were performed using the Orbitrap technology. The treatment of rmCK with SM resulted in a decrease of enzyme activity. However, this decrease did only weakly correlate to the modification of Cys283 but was conclusive for the formation of Met70-HETE and Met179-HETE. In contrast, the activity of mutants of rmCK produced by side-directed mutagenesis that contained substitutions of the respective Met residues (Met70Ala, Met179Leu, and Met70Ala/Met179Leu) was highly resistant against SM. Our results point to a critical role of the surface exposed Met70 and Met179 residues for CK activity.

Alkylated epidermal creatine kinase as a biomarker for sulfur mustard exposure: comparison to adducts of albumin and DNA in an in vivo rat study

Sulfur mustard (SM) is a chemical warfare agent which use is banned under international law and that has been used recently in Northern Iraq and Syria by the so-called Islamic State. SM induces the alkylation of endogenous proteins like albumin and hemoglobin thus forming covalent adducts that are targeted by bioanalytical methods for the verification of systemic poisoning. We herein report a novel biomarker, namely creatine kinase (CK) B-type, suitable as a local biomarker for SM exposure on the skin. Human and rat skin were proven to contain CK B-type by Western blot analysis. Following exposure to SM ex vivo, the CK-adduct was extracted from homogenates by immunomagnetic separation and proteolyzed afterwards. The cysteine residue Cys282 was found to be alkylated by the SM-specific hydroxyethylthioethyl (HETE)-moiety detected as the biomarker tetrapeptide TC(-HETE)PS. A selective and sensitive micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HRMS) method was developed to monitor local CK-adducts in an in vivo study with rats percutaneously exposed to SM. CK-adduct formation was compared to already established DNA- and systemic albumin biomarkers. CK- and DNA-adducts were successfully detected in biopsies of exposed rat skin as well as albumin-adducts in plasma. Relative biomarker concentrations make the CK-adduct highly appropriate as a local dermal biomarker. In summary, CK or rather Cys282 in CK B-type was identified as a new, additional dermal target of local SM exposures. To our knowledge, it is also the first time that HETE-albumin adducts, and HETE-DNA adducts were monitored simultaneously in an in vivo animal study.

Identification of creatine kinase and alpha-1 antitrypsin as protein targets of alkylation by sulfur mustard.

Sulfur mustard (SM) is a toxic chemical warfare agent deployed in several conflicts within the last 100 years and still represents a threat in terroristic attacks and warfare. SM research focuses on understanding the pathophysiology of SM and identifying novel biomarkers of exposure. SM is known to alkylate nucleophilic moieties of endogenous proteins, for example, free thiol groups of cysteine residues. The two-dimensional-thiol-differences in gel electrophoresis (2D-thiol-DIGE) technique is an initial proteomics approach to detect proteins with free cysteine residues. These amino acids are selectively labeled with infrared-maleimide dyes visualized after GE. Cysteine residues derivatized by alkylating agents are no longer accessible for the maleimide-thiol coupling resulting in the loss of the fluorescent signal of the corresponding protein. To prove the applicability of 2D-thiol-DIGE, this technology was exemplarily applied to neat human serum albumin treated with SM, to lysates from human cell culture exposed to SM as well as to human plasma exposed to CEES (chloroethyl ethyl sulfide, an SM analogue). Exemplarily, the most prominent proteins modified by SM were identified by matrix-assisted laser desorption/ionization time-of-flight (tandem) mass spectrometry, MALDI-TOF MS(/MS), as creatine kinase (CK) from human cells and as alpha-1 antitrypsin (A1AT) from plasma samples. Peptides containing the residue Cys282 of CK and Cys232 of A1AT were unambiguously identified by micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (μLC-ESI MS/HR MS) as being alkylated by SM bearing the specific hydroxyethylthioethyl-(HETE)-moiety. Both peptides might represent potential biomarkers of SM exposure. This is the first report introducing these endogenous proteins as targets of SM alkylation.

Quantitation by liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry of DNA adducts derived from methyl glyoxal and carboxyethylating agents in leukocytes of smokers and non-smokers

A liquid chromatograpy-nanoelectrospray ionization-high resolution tandem mass spectrometry (LC–NSI–HRMS/MS) method was developed for quantitation of the DNA adducts 7-(2′-carboxyethyl)guanine (7-2′-CEG) and N2-(1′-carboxyethyl)guanine (N2-1′-CEG), as their methyl esters, in human leukocyte DNA from smokers and non-smokers. 7-2′-CEG has been previously identified in all human liver samples analyzed and is formed from an unknown carboxyethylating agent while N2-1′-CEG is formed from the advanced glycation endproduct methyl glyoxal. The method was applied for the analysis of these two DNA adducts in leukocyte DNA from 20 smokers and 20 non-smokers, in part to test the hypothesis that 7-2′-CEG could be formed by endogenous nitrosation, as previously observed in rats treated with nitrosodihydrouracil and nitrite. Levels of 7-2′-CEG (mean ± S.D.) were 0.6 ± 0.2 pmol/μmol dG in smokers and 0.5 ± 0.2 pmol/μmol dG in non-smokers, while those of N2-1′-CEG were 4.5 ± 1.9 pmol/μmol dG in smokers and 4.6 ± 2 pmol/μmol dG in non-smokers. These results did not support our hypothesis that endogenous nitrosation of dihydrouracil in smokers leads to higher levels of 7-2′-CEG in leukocyte DNA than in non-smokers. However the study provides the first data on levels of these DNA adducts in human leukocyte DNA, and the LC–NSI–HRMS/MS method developed for their quantitation could be important for future studies of DNA damage by methyl glyoxal.

Total sulfur analysis of fine particulate mass on nylon filters by ICP-OES.

Sulfur (S) and sulfate (SO4 2- ) in fine particulate matter (PM2.5 ) are monitored by the Interagency Monitoring of Protected Visual Environments (IMPROVE) network at remote and rural sites across the United States. Within the IMPROVE network, S is determined from X-ray fluorescence (XRF) spectroscopy from a Teflon filter, and SO4 2- is determined via ion chromatography (IC) from a nylon filter. Differences in S and SO4 2- estimates may indicate the presence of organosulfur (OS) species or biases between sampling and analytical methods. To reduce potential biases, an inductively coupled plasma-optical emission spectroscopy (ICP-OES) method was developed to allow for analysis of SO4 2- and S from a single filter extract. Sulfur (ICP-OES) and SO4 2- (IC) estimates from 2016 IMPROVE filters correlated strongly, suggesting that, on average, ICP-OES accurately estimated S. However, observed differences between slopes suggested the presence of water-soluble OS species, especially during summer. Organosulfur species are important indicators of secondary organic aerosols formed through reactions of biogenic and anthropogenic pollutants and can be quantified through laboratory techniques such as reverse-phase liquid chromatography (RPLC) or hydrophilic liquid interaction chromatography (HILIC) coupled to electrospray ionization-high-resolution tandem mass spectrometry (RPLC/ESI-HR-MS/MS and HILIC/ESI-HR-MS/MS, respectively), and field techniques using Aerodyne aerosol mass spectrometry (AMS). However, these methods are costly and introduce relatively large uncertainties when scaled for large networks such as IMPROVE. The method described in this report provides an inexpensive complement to XRF, which measures total S (insoluble and water-soluble S) to estimate water-soluble S and OS concentrations in PM.

Investigation of Fragmentation Pathways of Fentanyl Analogues and Novel Synthetic Opioids by Electron Ionization High-Resolution Mass Spectrometry and Electrospray Ionization High-Resolution Tandem Mass Spectrometry.

The global drug market is characterized by the fast development of new psychoactive substances such as fentanyl analogues and novel synthetic opioids, the detection of which is complicated by the lack of appropriate quality control procedures and references. Herein, we analyze the fragmentation pathways and characteristic ions of 25 novel fentanyl analogues and 5 novel synthetic opioids by electron ionization (EI) and electrospray ionization (ESI) high-resolution mass spectrometry to provide a reference for the identification of these species. In the ESI mode, fentanyl analogues mainly undergo piperidine ring degradation, phenethyl and piperidine ring dissociation, and piperidine ring and amide moiety cleavage, while piperidine ring degradation and phenethyl and piperidine ring dissociation are the major pathways in the EI mode. The five novel synthetic opioids largely undergo amide group dissociation and N-cyclohexyl bond cleavage in the ESI mode. Thus, this work facilitates the detection and quantitation of fentanyl analogues and novel synthetic opioids or other substances with similar structures in forensic laboratories.

Cytokinin Detection during the Dictyostelium discoideum Life Cycle: Profiles Are Dynamic and Affect Cell Growth and Spore Germination.

Cytokinins (CKs) are a family of evolutionarily conserved growth regulating hormones. While CKs are well-characterized in plant systems, these N6-substituted adenine derivatives are found in a variety of organisms beyond plants, including bacteria, fungi, mammals, and the social amoeba, Dictyostelium discoideum. Within Dictyostelium, CKs have only been studied in the late developmental stages of the life cycle, where they promote spore encapsulation and dormancy. In this study, we used ultra high-performance liquid chromatography-positive electrospray ionization-high resolution tandem mass spectrometry (UHPLC-(ESI+)-HRMS/MS) to profile CKs during the Dictyostelium life cycle: growth, aggregation, mound, slug, fruiting body, and germination. Comprehensive profiling revealed that Dictyostelium produces 6 CK forms (cis-Zeatin (cZ), discadenine (DA), N6-isopentenyladenine (iP), N6-isopentenyladenine-9-riboside (iPR), N6-isopentenyladenine-9-riboside-5′ phosphate (iPRP), and 2-methylthio-N6-isopentenyladenine (2MeSiP)) in varying abundance across the sampled life cycle stages, thus laying the foundation for the CK biosynthesis pathway to be defined in this organism. Interestingly, iP-type CKs were the most dominant CK analytes detected during growth and aggregation. Exogenous treatment of AX3 cells with various CK types revealed that iP was the only CK to promote the proliferation of cells in culture. In support of previous studies, metabolomics data revealed that DA is one of the most significantly upregulated small molecules during Dictyostelium development, and our data indicates that total CK levels are highest during germination. While much remains to be explored in Dictyostelium, this research offers new insight into the nature of CK biosynthesis, secretion, and function during Dictyostelium growth, development, and spore germination.

Forensic evidence of sulfur mustard exposure in real cases of human poisoning by detection of diverse albumin-derived protein adducts.

We present the forensic analyses of plasma samples of human victims exposed to sulfur mustard (SM) in a crisis region in the Middle East in 2015. A few hours after exposure, poisoned persons showed typical signs and symptoms of percutaneous SM exposure including erythema and later on blisters and hardly healing skin wounds. Blood samples were collected 15days after poisoning to be analyzed for the presence of long-lived protein-adduct biomarkers to verify SM poisoning. We applied a novel bioanalytical toolbox targeting four human serum albumin-derived biomarkers that were made accessible after plasma proteolysis. These adducts contained the SM-specific hydroxyethylthioethyl moiety either bound to the thiol group of a cysteine residue (C34*) or to the side-chain carboxylic group of a glutamic acid residue (E230*). Peptide biomarkers were produced from plasma of the victims using proteinase K (C34*PF), pronase (C34*P) and pepsin (AE230*VSKL and LQQC34*PFEDHVKL) for enzymatic protein cleavage. Separation and detection were carried out by selective micro-liquid chromatography-electrospray ionization high-resolution tandem mass spectrometry (µLC-ESI MS/HR MS). In addition to this site-specific adduct detection, a general approach after alkaline hydrolysis of the plasma protein fraction was applied. Liberated thiodiglycol (TDG) was derivatized with heptafluorobutyric anhydride and detected by gas chromatography-electron ionization mass spectrometry (GC-EI MS). The different bioanalytical methods yielded congruent results confirming SM poisoning for all patients who showed clinical signs and symptoms. This is the first time that real cases of SM poisoning were confirmed and presented by such a broad compilation of protein-derived biomarkers.

Methyl DNA phosphate adduct formation in lung tumor tissue and adjacent normal tissue of lung cancer patients.

The formation of methyl DNA adducts is a critical step in carcinogenesis initiated by the exposure to methylating carcinogens. Methyl DNA phosphate adducts, formed by methylation of the oxygen atoms of the DNA phosphate backbone, have been detected in animals treated with methylating carcinogens. However, detection of these adducts in human tissues has not been reported. We developed an ultrasensitive liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method for detecting methyl DNA phosphate adducts. Using 50 μg of human lung DNA, a limit of quantitation of two adducts/1010 nucleobases was achieved. Twenty-two structurally unique methyl DNA phosphate adducts were detected in human lung DNA. The adduct levels were measured in both tumor and adjacent normal tissues from 30 patients with lung cancer, including 13 current smokers and 17 current non-smokers, as confirmed by measurements of urinary cotinine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Levels of total methyl DNA phosphate adducts in normal lung tissues were higher in smokers than non-smokers, with an average of 13 and 8 adducts/109 nucleobases, respectively. Methyl DNA phosphate adducts were also detected in lung tissues from untreated rats with steady-state levels of 5-7 adducts/109 nucleobases over a period of 70 weeks. This is the first study to report the detection of methyl DNA phosphate adducts in human lung tissues. The results provide new insights toward using these DNA adducts as potential biomarkers to study human exposure to environmental methylating carcinogens.

Matrix-induced transformation of arsenic species in seafoods

The paper presents a study on the matrix-induced transformation of arsenic (As) species spiked into seafoods. Sixteen arsenicals were individually spiked into samples of finfish, crustaceans and molluscs. The spiked samples were subjected to hot water extraction at 90 °C, and extracts were analyzed by high pressure liquid chromatography (HPLC) interfaced with inductively coupled plasma mass spectrometry (ICP-MS). Arsenate (As5+), arsenobetaine (AsB), arsenocholine (AsC), dimethylarsinate (DMA), monomethylarsonate (MMA), tetramethylarsonium ion (TMA) and trimethylarsoniopropionate (TMAP) remained intact in all the matrices. Whereas arsenite (As3+), dimethylarsinoyl acetate (DMAA), dimethylarsinoyl ethanol (DMAE), dimethylarsinoyl propionate (DMAP), trimethylarsine oxide (TMAO) and glycerol-, sulfonate-, sulfate- and phosphate-arsinoylribosides (arsenosugars 328, 392, 408 and 482, respectively) were transformed to other forms in most finfish and crustaceans. The transformation of the arsenicals was discovered to be induced by matrix thiols. While As3+ was bound to sulfhydryl groups, DMAA, DMAE, DMAP, TMAO and arsenosugars 392, 408 and 482 were thiolated through conversion of their arsinoyl (As=O) functionalities to arsinothioyl (As=S). The newly formed arsinothioyl compounds were characterized by HPLC-ICP-MS and electrospray ionization high-resolution tandem mass spectrometry (ESI-HR-MS/MS) paired with HPLC. The observed matrix-induced transformation of the arsenic species could be prevented by treating the samples (prior to spiking) with a thiol-selective blocking agent, N-ethylmaleimide (NEM).