Ionization High-resolution Tandem Mass Research Articles

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.

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.

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.

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).

Isolation of high-purity peptide Val-Val-Tyr-Pro from Globin Peptide using MCI gel column combined with high-speed counter-current chromatography.

Peptides have gained increased interest over the past several decades because of their therapeutics. In this research, a strategy combining MCI gel column chromatography and high-speed countercurrent chromatography was developed for the separation of high-purity peptide Val-Val-Tyr-Pro from Globin Peptide. First, the fraction of Val-Val-Tyr-Pro mixtures with a purity of 15.8% was obtained by using MCI gel column with a mixture of ethanol/water (20:80, v/v/v). Then, the high-purity Val-Val-Tyr-Pro was separated by high-speed countercurrent chromatography with a aqueous two phase systems of ethanol/acetonitrile/iso-propyl alcohol/(NH4 )2 SO4 Saturated solution /H2 O (0.5:0.5:0.25:1.5:0.7,v/v). The ammonium sulfate from high-speed countercurrent chromatography fractions was removed from target compound by MCI gel column chromatography using ethanol/water in stepwise elution mode. A 78mg of Val-Val-Tyr-Pro was successfully purified with the purities of 98.80% from 30g crude Globin Peptide. The amino acid sequence of the Val-Val-Tyr-Pro was determined by electrospray ionization high resolution tandem mass spectrometry. The method presents a practical strategy for the large-scale separation of pure peptide Val-Val-Tyr-Pro from Globin Peptide, and provides a reference method for obtaining high-purity peptide from other polypeptide mixtures.

Identification and structural characterization of in vivo metabolites of balofloxacin in rat plasma, urine and feces samples using Q-TOF/LC/ESI/MS/MS : In silico toxicity studies

Balofloxacin is a fluroquinolone antibiotic drug which has been used for the treatment of urinary tract infections (UTIs). Identification and structural characterization of metabolites is a critical component of both drug discovery and drug development research. In vivo metabolites of balofloxacin have been identified and characterized by using liquid chromatography positive ion electrospray ionization high resolution tandem mass spectrometry (LC/ESI-HR-MS/MS) experiments. To identify in vivo metabolites, blood, urine and feces samples were collected after oral administration of the drug to the female Sprague-Dawley rats (n = 3 per group). Protein precipitation, freeze liquid separation followed by solid-phase extraction methods were used for sample preparation. The extracted samples were subjected to LC-ESI/HRMS/MS analysis. The chromatographic separation of the drug and its metabolites were achieved on a XDB, C18 (50, 4.6 mm, 5 mm) column using gradient elution method in combination with 0.1% formic acid and acetonitrile at a flow rate of 0.4 mL/min. A total of 13 phase I and phase II metabolites of balofloxacin have been identified in plasma, urine and feces samples. Most of metabolites were observed in plasma and urine samples including dealkylated, desmethylated, decarbonylated, decarboxylated, hydroxylated, methylated, carboxylated, cysteine conjugated metabolites and high abundance glucuronidated metabolite. The structures of metabolites have been elucidated based on fragmentation patterns, accurate mass measurements and LC/MS/MS experiments. The main phase I metabolites of balofloxacin, decarbonylated, decarboxylated and desmethylated metabolites and phase II methylated metabolite undergo subsequent phase II glucuronidation pathways. In silico toxicity of the drug and its metabolites was determined using ProTox-II. Metabolites B-1, B-2, B-5, B-6, B-7, and B-8 to B-13 were predicted to possess immunotoxicity with high probability score. Additionally, Amine Oxidase A and Prostaglandin G/H Synthase 1 are predicted for metabolites B-1, B-3 to B-6 as toxicity targets with binding probability.

Trace analysis of multi-class phytohormones in Oryza sativa using different scan modes in high-resolution Orbitrap mass spectrometry: method validation, concentration levels, and screening in multiple accessions.

Phytohormones are signaling and regulating metabolites involved in numerous plant processes, including growth, development, and responses to stress. Currently, the focus is on the analysis of multiple phytohormones in order to characterize crosstalk and hormone signaling networks. In this paper, representative phytohormones of the major classes are simultaneously determined in rice tissues by a generic solid-liquid extraction, followed by liquid chromatography and electrospray ionization high-resolution tandem mass spectrometry using a Q-Exactive™ instrument. After a thorough optimization of the sample preparation, the analytical method was fully validated toward the ultra-trace quantification of six a priori selected plant hormones using three scan modes of the quadrupole-Orbitrap instrument: full-scan high-resolution mass spectrometry, targeted single ion monitoring (t-SIM), and t-SIM followed by data-dependent tandem mass spectrometry. Overall, a similar quantitative performance was noticed for the different scan modes. The analytical method was successfully applied to measure basal phytohormone levels in six different rice accessions, comprising Oryza sativa ssp. japonica, indica, and Oryza glaberrima. Hormone concentrations were higher in shoots than in roots or at least similar. Except for a lower level of salicylic acid in shoots of O. glaberrima versus O. sativa, no other differences in hormone levels could be noticed that were dependent of the (sub)species assignment of the analyzed accessions. Making use of the benefits of full-scan high-resolution mass spectrometry, a first post-run suspect screening was performed, suggesting - based on accurate mass measurements and isotopic patterns - the possible presence of about 50 additional plant hormones in the rice tissues. Graphical abstract ᅟ.

Methyl DNA Phosphate Adduct Formation in Rats Treated Chronically with 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of Its Metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a powerful lung carcinogen in animal models and is considered a causative factor for lung cancer in tobacco users. NNK is stereoselectively and reversibly metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is also a lung carcinogen. Both NNK and NNAL undergo metabolic activation by α-hydroxylation on their methyl groups to form pyridyloxobutyl and pyridylhydroxybutyl DNA base and phosphate adducts, respectively. α-Hydroxylation also occurs on the α-methylene carbons of NNK and NNAL to produce methane diazohydroxide, which reacts with DNA to form methyl DNA base adducts. DNA adducts of NNK and NNAL are important in their mechanisms of carcinogenesis. In this study, we characterized and quantified methyl DNA phosphate adducts in the lung of rats treated with 5 ppm of NNK, (S)-NNAL, or (R)-NNAL in drinking water for 10, 30, 50, and 70 weeks, by using a novel liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method. A total of 23, 21, and 22 out of 32 possible methyl DNA phosphate adducts were detected in the lung tissues of rats treated with NNK, (S)-NNAL, and (R)-NNAL, respectively. Levels of the methyl DNA phosphate adducts were 2290–4510, 872–1120, and 763–1430 fmol/mg DNA, accounting for 15–38%, 8%, and 5–9% of the total measured DNA adducts in rats treated with NNK, (S)-NNAL, and (R)-NNAL, respectively. The methyl DNA phosphate adducts characterized in this study further enriched the diversity of DNA adducts formed by NNK and NNAL. These results provide important new data regarding NNK- and NNAL-derived DNA damage and new insights pertinent to future mechanistic and biomonitoring studies of NNK, NNAL, and other chemical methylating agents.

Speciation of CuCl and CuCl2 Thiol-Amine Solutions and Characterization of Resulting Films: Implications for Semiconductor Device Fabrication.

Thiol-amine mixtures are an attractive medium for the solution processing of semiconducting thin films because of their remarkable ability to dissolve a variety of metals, metal chalcogenides, metal salts, and chalcogens. However, very little is known about their dissolution chemistry. Electrospray ionization high-resolution tandem mass spectrometry and X-ray absorption spectroscopy were employed to identify the species formed upon dissolution of CuCl and CuCl2 in 1-propanethiol and n-butylamine. Copper was found to be present exclusively in the 1+ oxidation state for both solutions. The copper complexes detected include copper chlorides, copper thiolates, and copper thiolate chlorides. No complexes of copper with amines were observed. Additionally, alkylammonium ions and alkylammonium chloride adducts were observed. These findings suggest that the dissolution is initiated by proton transfer from the thiol to the amine, followed by coordination of the thiolate anions with copper cations. Interestingly, the mass and X-ray absorption spectra of the solutions of CuCl and CuCl2 in thiol-amine were essentially identical. However, dialkyl disulfides were identified by Raman spectroscopy as an oxidation product only for the copper(II) solution, wherein copper(II) had been reduced to copper(I). Analysis of several thiol-amine pairs suggested that the dissolution mechanism is quite general. Finally, analysis of thin films prepared from these solutions revealed persistent chlorine impurities, in agreement with previous studies. These impurities are explained by the mass spectrometric finding that chloride ligands are not completely displaced by thiolates upon dissolution. These results suggest that precursors other than chlorides will likely be preferred for the generation of high-efficiency copper chalcogenide films, despite the reasonable efficiencies that have been obtained for films generated from chloride precursors in the past.

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s.

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted mechanism of metabolic activation involves formation of α-hydroxynitrosamines that spontaneously decompose to DNA alkylating agents. Accumulation of DNA damage and the resulting mutations can ultimately lead to cancer. New evidence indicates that α-hydroxynitrosamines can be further oxidized to nitrosamides processively by cytochrome P450s. Because nitrosamides are generally more stable than α-hydroxynitrosamines and can also alkylate DNA, nitrosamides may play a role in carcinogenesis. In this report, we describe a general protocol for evaluating nitrosamide production from in vitro cytochrome P450-catalyzed metabolism of nitrosamines. This protocol utilizes a general approach to the synthesis of the relevant nitrosamides and an in vitro cytochrome P450 metabolism assay using liquid chromatography-nanospray ionization-high resolution tandem mass spectrometry for detection. This method detected N'-nitrosonorcotinine as a minor metabolite of N'-nitrosonornicotine in the example study. The method has high sensitivity and selectively due to accurate mass detection. Application of this method to a wide variety of nitrosamine-cytochrome P450 systems will help determine the generality of this transformation. Because cytochrome P450s are polymorphic and vary in activity, a better understanding of nitrosamide formation could aid in individual cancer risk assessment.

Investigation of the presence in human urine of mercapturic acids derived from phenanthrene, a representative polycyclic aromatic hydrocarbon

Polycyclic aromatic hydrocarbons (PAH) are environmental carcinogens implicated as causes of cancer in certain industrial settings and in cigarette smokers. PAH require metabolic activation to exert their carcinogenic effects. One widely accepted pathway of metabolic activation proceeds through formation of “bay region” diol epoxides which are highly reactive with DNA and can cause mutations. Phenanthrene (Phe) is the simplest PAH with a bay region and an excellent model for the study of PAH metabolism. In previous studies in which [D10]Phe was administered to smokers, we observed higher levels of [D10]Phe-tetraols derived from [D10]Phe-diol epoxides in subjects who were null for the glutathione-S-transferase M1 (GSTM1) gene. We hypothesized that Phe-epoxides, the primary metabolites of Phe, were detoxified by glutathione conjugate formation, which would result ultimately in the excretion of the corresponding mercapturic acids in urine. We synthesized the four stereoisomeric mercapturic acids that would result from attack of glutathione on Phe-epoxides followed by normal processing of the conjugates. We also synthesized the corresponding dehydrated metabolites and sulfoxides. These 12 standards were used in liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry analysis of urine samples from smokers and creosote workers, the latter exposed to unusually high levels of PAH. Only the sulfoxide derivatives were consistently detected in the urine of creosote workers; none of the compounds was detected in the urine of smokers. These results demonstrate a new pathway of PAH-mercapturic acid formation, but do not provide an explanation for the role of GSTM1 null status on Phe-tetraol formation.