Gas Chromatography-electron Ionization-mass Spectrometry Research Articles

Metabolomic Profiling of Biological Reference Materials using a Multiplatform High-Resolution Mass Spectrometric Approach.

The number of metabolomics studies have increased dramatically in recent years, spanning from basic/mechanistic research to the identification and validation of clinical biomarkers. Developments in analyte separation techniques and the growth of databases are largely responsible for the rapid growth of metabolomics, although broad differences in analytical workflows can result in difficulty when comparing data across studies. The establishment of baseline metabolomics data for human reference materials using complementary/orthogonal data acquisition strategies can help to alleviate some of these challenges. To this end, we report nontargeted semiquantitative metabolomics data for 22 commercially available materials including plasma (healthy, diabetic, hypertriglyceridemic, African-American), serum (female, male, pregnant, among others), feces (meconium, vegan, omnivore), urine (smokers' and nonsmokers'), breast milk, saliva, and vaginal fluid, using ultrahigh-performance liquid chromatography-tandem mass spectrometry in positive and negative electrospray ionization, as well as gas chromatography-electron ionization-mass spectrometry. Significant differences were observed in the metabolomic fingerprints between all sample types. Post hoc comparisons between relevant sample types support the relevance of these materials and the validity of nontargeted strategies in global metabolomics. As the number and variety of reference materials continues to increase, it is imperative that their adoption is matched. The results of this study may inform future biomedical research by highlighting several metabolites across matrixes and treatments/states that could serve as clinical biomarkers or important biochemical pathway intermediates. Furthermore, our work can serve as a metric for systems suitability, quality assurance, and quality control across the community via the dissemination of high-quality and publicly available annotated metabolomics data.

Trocylation of 3-quinuclidinol, a key marker for the chemical warfare agent 3-quinuclidinyl benzilate, for its enhanced detection at low levels in complex soil matrices by electron ionization gas chromatography-mass spectrometry.

Detection of 3-quinuclidinol (3Q), a marker for the chemical warfare agent 3-quinuclidinyl benzilate, is very difficult by gas chromatography-mass spectrometry (GC/MS), providing low, broad signals even when analyzed in isolated form. Therefore, a method that can convert 3Q into a substrate with enhanced detectability by GC/MS would be an important tool for its analysis. 2,2,2-Trichloroethoxycarbonyl chloride (TrocCl) was used in the derivatization of 3Q in three different soils of varying composition and total organic content (Virginia type A soil, Nebraska EPA standard soil and Ottawa sand) when present at a 10μg g-1 concentration in each. A direct derivatization protocol and one involving the pre-extraction of the analyte were evaluated for their individual efficiencies and subsequent analysis using electron ionization GC/MS. The practical derivatization of 3Q, when present at low levels (10μg g-1 ) in three different soil matrices, was found to be rapid (1h) and to take place smoothly at ambient temperature (and as low as 4°C). The method detection limit was determined to be 30 ng mL-1 for the Virginia type A soil, 49 ng mL-1 for the Nebraska EPA standard soil and 72 ng mL-1 for the Ottawa sand sample. An expedient and practical derivatization method for 3Q, a chemical warfare degradation product difficult to detect by GC/MS, has been realized using trichloroethyl chloroformate. The reaction provides 3Q-Troc, a derivative with better detectability than 3Q by electron ionization GC/MS such as peak sharpness and a unique mass spectrum for its unambiguous identification.

Two Food Waste By-Products Selectively Stimulate Beneficial Resident Citrus Host-Associated Microbes in a Zero-Runoff Indoor Plant Production System

The global production of food waste is a far-reaching problem with sizable financial, ethical, social, and environmental costs. Over 66 million tons of food waste is produced annually in the United States alone. This waste can be converted into valuable digestate by-products that promote a circular economy within agri-food systems. The present work investigated the use of two liquid digestates of microaerobic fermentation from mixed food waste and beer mash, respectively, as biostimulants for non-bearing citrus plants (nursery stock) grown in a zero-runoff greenhouse system with recirculating irrigation. The digestates' impact on the structure and diversity of the microbiota was determined on the irrigation water, soil, leaves, roots, and rhizosphere of citrus plants. A combination of culture-dependent (selective media) and culture-independent approaches (Next-Generation Sequencing) was used to assess the composition of the microbial communities and to single out the presence of foodborne pathogens. Our results suggest that the use of digestates is safe (i.e., no human or plant pathogens were present in the digestates or enriched in the plant production system following amendments). Digestates application to the irrigation water reduced the bacterial diversity within 24–48 h and selectively and significantly stimulated beneficial resident host-associated microorganisms (Pseudomonas putida) by two to three orders of magnitude. Carbon dynamics were analyzed in the nutrient solutions by measuring dissolved organic carbon and characterizing carbon species through gas chromatography-electron ionization-mass spectrometry. Our results indicate that dissolved organic carbon in the recirculating irrigation water spikes after each digestate amendment and it is quickly metabolized by bacteria, plateauing 24 h after application. Soil carbon, nitrogen, and nutrient dynamics were also analyzed, and results suggest that digestates increased the concentration of some plant nutrients in soils without causing a surge of potentially toxic elements. This study represents a proof-of-concept for the safe re-use of organic wastes, from farming and consumers, in agriculture. Implementing this type of integrated plant production system could reduce the environmental impact of food waste and benefit the public by improving soil health, reducing agricultural footprint, and increasing crop fitness by deploying a method based on a circular economy and sustainable food production approaches.

In‐depth gas chromatography/tandem mass spectrometry fragmentation analysis of formestane and evaluation of mass spectral discrimination of isomeric 3‐keto‐4‐ene hydroxy steroids

The aromatase inhibitor formestane (4-hydroxyandrost-4-ene-3,17-dione) is included in the World Anti-Doping Agency's List of Prohibited Substances in Sport. However, it also occurs endogenously as do its 2-, 6- and 11-hydroxy isomers. The aim of this study is to distinguish the different isomers using gas chromatography/electron ionization mass spectrometry (GC/EI-MS) for enhanced confidence in detection and selectivity for determination. Established derivatization protocols to introduce [2 H9 ]TMS were followed to generate perdeuterotrimethylsilylated and mixed deuterated derivatives for nine different hydroxy steroids, all with 3-keto-4-ene structure. Formestane was additionally labelled with H2 18 O to obtain derivatives doubly labelled with [2 H9 ]TMS and 18 O. GC/EI-MS spectra of labelled and unlabelled TMS derivatives were compared. Proposals for the generation of fragment ions were substantiated by high-resolution MS (GC/QTOFMS) and tandem mass spectrometry (MS/MS) experiments. Subclass-specific fragment ions include m/z 319 for the 6-hydroxy and m/z 219 for the 11-hydroxy compounds. Ions at m/z 415, 356, 341, 313, 269 and 267 were indicative for the 2- and 4-hydroxy compounds. For their discrimination the transition m/z 503 → 269 was selective for formestane. In 2-, 4- and 6-hydroxy steroids loss of a TMSO radical takes place as cleavage of a TMS-derived methyl radical and a neutral loss of (CH3 )2 SiO. Further common fragments were also elucidated. With the help of stable isotope labelling, the structures of postulated diagnostic fragment ions for the different steroidal subclasses were elucidated. 18 O-labelling of the other compounds will be addressed in future studies to substantiate the obtained findings. To increase method sensitivity MS3 may be suitable in future bioanalytical applications requiring discrimination of the 2- and 4-hydroxy compounds.

Electron ionization mass spectrometric fragmentation and detection of autoxidation products of 2,6,10,14-tetramethyl-7-(3-methylpent-4-enyl)-pentadec-5-ene in Arctic sediments.

Some highly branched isoprenoid (HBI) alkenes are commonly used as proxies for palaeoceanographic reconstructions. However, there is a need to identify compounds that are sufficiently stable and abundant to be used as tracers of HBI oxidation in sediments. 2,6,10,14-Tetramethyl-7-(3-methylpent-4-enyl)-pentadec-5(Z/E)-en-4-ols resulting from 2,6,10,14-tetramethyl-7-(3-methylpent-4-enyl)-pentadec-5-ene appear to be useful for this purpose. Comparison of electron ionization (EI) mass spectra and retention times with those of standards allowed formal identification of autoxidation products of 2,6,10,14-tetramethyl-7-(3-methylpent-4-enyl)-pentadec-5-ene. EI-MS fragmentations of TMS ethers of the main oxidation products (2,6,10,14-tetramethyl-7-(3-methylpent-4-enyl)-pentadec-5(Z/E)-en-4-ols) were deduced by gas chromatography/electron ionization mass spectrometry (GC/EI-MS), low-energy collision-induced dissociation tandem mass spectrometry (CID-MS/MS) and accurate mass measurements. These compounds were then quantified in Arctic sediment samples in MS/MS multiple reaction monitoring (MRM) mode using transitions based on the main fragmentation pathways elucidated. 2,6,10,14-Tetramethyl-7-(3-methylpent-4-enyl)-pentadec-5(Z/E)-en-4-ols were identified after autoxidation of the HBI alkene 2,6,10,14-tetramethyl-7-(3-methylpent-4-enyl)-pentadec-5-ene. Low-energy CID-MS/MS analyses and accurate mass measurements allowed the EI-MS fragmentation pathways of their trimethylsilyl (TMS) derivatives to be elucidated. Some specific fragment ions and chromatographic retention times were also useful for further characterization. As an application of some of the described fragmentations, TMS derivatives of these metabolites were characterized and quantified in MRM mode in Arctic sediments. Due to their production in high proportions during autoxidation of their parent HBI diene, their apparent stability in sediments, and their specific EIMS fragmentations, (Z and E)-2,6,10,14-tetramethyl-7-(3-methylpent-4-enyl)-pentadec-5-en-4-ol TMS derivatives appeared to be useful tracers of HBI autoxidation in sediments.

Impossible to Go Beyond Beef? An Unbiased Metabolomics Analysis

Abstract Objectives Novel plant-based meat alternatives are becoming increasingly popular with consumers. The new generation of meat alternatives is specifically formulated to closely mimic the taste, sensory experience, and nutritional composition of meat. The goal of this work was to perform unbiased analysis of the biochemical composition of ground beef versus a novel plant-based meat alternative to determine their nutritional equivalency. Methods Ground beef from eighteen different steers was purchased from Alderspring Ranch, ID. Eighteen different packages of a plant-based meat alternative were purchased from a local grocery store. Non-targeted metabolomic analysis of sample homogenates was conducted via gas chromatography/electron-ionization mass spectrometry (GC/ei-MS) in the Metabolomics Laboratory of the Duke Molecular Physiology Institute. Following log-transformation, t-test were used to compare all individual analytes using a p-value (false discovery rate) cutoff of P < 0.01, without adjustment for multiple comparisons. Multivariate analyses (principal component and cluster analysis) was performed using software procedures in MetaboAnalyst 4.0. Results A total of 159 out of 184 detected metabolites were found to be different between beef and the plant-based meat alternative (P < 0.01). Metabolites such as niacinamide, calcium pantothenate, creatinine/creatine, anserine, hydroxyproline, and glucosamine were only found in beef, while metabolites such as delta-tocopherol, 3-hydroxyanthranilic acid, vanillic acid, and various plant sterols were only found in the plant-based alternative. Conclusions Despite suggested similarity based on their Nutrition Facts panel, we show that metabolites with important regulatory roles in human health are either absent or present in lower quantities in the plant-based meat alternative. These data suggest that novel plant-based meat alternatives should, at present, not be viewed as direct nutritional replacements for red meat. Funding Sources None.

Fragmentation pathways of odd- and even-electron N-alkylated synthetic cathinones

Three ionization techniques, isotopic labeling, high-resolution mass spectrometry (HRMS) and multi-stage mass spectrometry (MSn) were used to analyze a series of N-alkylated synthetic cathinone derivatives and gain a deeper understanding of their fragmentation behavior during mass spectrometric analysis. The compounds analyzed represent 15 unique structures with common substitutions to the core synthetic cathinone structure, including substitutions to the aromatic ring and the number and types of N-alkyl functionalities. The analytical techniques employed include gas chromatography-electron ionization-mass spectrometry (GC-EI-MS), electrospray ionization-tandem mass spectrometry (ESI-MS/MS) with HRMS and direct analysis in real time tandem mass spectrometry (DART-MS/MS) with HRMS. These techniques cover a variety of forensic applications, including seized drug analysis, toxicological analysis and screening analysis, in local, state and federal laboratories. For collision-induced dissociation (CID) of protonated precursors, the spectra of 2° and 3° amines showed evidence for charge-remote and charge-directed fragmentation mechanisms. The 2° amines lost H2O as a dominant pathway whereas the 3° amines favored the formation of alkylphenones. As reported by others, CID of protonated N-alkylated cathinones containing 2° and 3°amines also provided an abundance of odd-electron product ions from the even-electron precursors. In contrast to the rearrangements observed in CID of protonated cathinones, EI fragmentation patterns were dominated by radical-directed cleavages to form iminium ions and charge-directed cleavages to form acylium ions. A comparison between the fragmentation behaviors of N-alkylated synthetic cathinones under all three ionization techniques enables a deeper understanding of N-alkylated synthetic cathinone fragmentation under varying instrumental setups.

Mass spectral fragmentation of perfluoroacyl derivatives of half nitrogen mustards for their detection by gas chromatography/mass spectrometry.

Analytical methods for the detection and identification of half nitrogen mustards (halfNMs), i.e., partially hydrolyzed products of nitrogen mustards (pHpNMs), using silyl derivatives are often associated with low sensitivity and selectivity. In order to overcome these limitations, the derivatization of halfNMs was performed using perfluoroacylation. Two efficient derivatization techniques using trifluoroacetyl (TFA) and heptafluorobutyryl (HFB) groups were developed for the unambiguous identification of halfNMs. A mass spectral database was generated by performing gas chromatography/electron ionization mass spectrometry (GC/EI-MS) and gas chromatography/positive chemical ionization mass spectrometry (GC/PCI-MS). The fragmentation pathways were studied by tandem mass spectrometry (MS/MS) in both EI and PCI mode. The EI-MS spectra of the TFA and HFB derivatives of halfNMs contain intense molecular ions and fragment ions, thus making perfluoroacylation preferable to silylation. In addition, the background-free chromatogram obtained using these derivatives provides unambiguous identification of these compounds in blind samples. The structures of the fragment ions were postulated, and the sources of significant ions were traced by performing MS/MS precursor ion scans. In the PCI-MS spectra, along with the protonated molecule, significant peaks due to neutral losses of HF, HCl, CH3 Cl and CF3 COOH were observed. This is the first report of the elucidation of the fragmentation pathways of perfluoroacyl derivatives of halfNMs. The complementary GC/PCI-MS and GC/PCI-MS/MS data will be helpful in the identification of unknown metabolites in a fast and reliable fashion.

Fragmentation pathways of α-pyrrolidinophenone synthetic cathinones and their application to the identification of emerging synthetic cathinone derivatives

The expanding use of emerging synthetic drugs is creating a growing problem for both seized drug analysts and toxicologists because the clandestine suppliers continually tweak the chemical structures to keep one step ahead of the law. Synthetic cathinones, commonly referred to as bath salts, are a specific class of emerging synthetic drugs. These substances are derivatives of cathinone, which is the psychoactive component of the Catha edulis plant, commonly referred to as khat. Of the synthetic cathinone class of compounds, the α-pyrrolidinophenone synthetic cathinone derivatives stand out as one of the most abused designer drugs.The fragmentation behavior of a series of α-pyrrolidinophenone synthetic cathinones was studied with three different ionization and fragmentation techniques to enhance the current understanding of α-pyrrolidinophenone synthetic cathinones in mass spectrometers. Gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) fragmentation is commonly used by seized drug analysts, whereas liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is more commonly used in toxicological analyses. Direct analysis in real time mass spectrometry (DART-MS) is becoming more popular as a screening technique, especially in national laboratories. Each ionization and activation method encourage particular pathways of fragmentation, and whereas some pathways are conserved across all platforms, other pathways are unique to a particular instrument. This study combines isotope-labeling, multi-stage mass spectrometry (MSn) and accurate mass measurements with high-resolution mass spectrometry (HRMS) to enhance the current understanding about α-pyrrolidinophenone synthetic cathinones. This manuscript provides characteristic protonated tandem mass spectrometry fragmentation pathways and the mechanistic origins of the EI-MS fragmentation observed for this class of synthetic cathinones and provides examples of how this knowledge can be applied to the identification of novel synthetic cathinones.

A simplified method for therapeutic drug monitoring of mitotane by gas chromatography-electron ionization-mass spectrometry.

Mitotane is a key drug for the treatment of adrenal cortical carcinoma. Due to its narrow therapeutic window, 14–20 μg/mL, monitoring its concentration is crucially important. In this study, a simplified method for measuring mitotane in plasma using gas chromatography‐electron ionization‐mass spectrometry (GC‐EI‐MS) was developed. Through deproteination and liquid–liquid extraction, mitotane and an internal standard (IS) were extracted from plasma samples. GC‐EI‐MS yielded retention times of 8.2 and 8.7 min, for mitotane and the IS, respectively, with a total run time of 12 min. Selectivity and intra‐/inter‐batch accuracy and precision analyses provided a lower limit of quantification of 0.25 μg/mL, and a calibration curve between 0.25 and 40 μg/mL had good linearity (coefficient of determination = 0.992). The matrix effect factor and percent recovery of the method had good precision. Additionally, long‐term sample stability was observed below 4°C. In a clinical setting, mitotane levels in plasma from an adrenal cortical carcinoma patient were within calibration range. Therefore, this simplified method can be applied to routine therapeutic drug monitoring of mitotane, which may contribute to improved treatment of adrenal cortical carcinoma.

Biological Activity and Composition of the Essential Oil and Fatty Constituents of Petroleum ether Extract of Brassica juncea (L.)

Brassica juncea L. (Brassicaceae) is widely used in making mustard oil. The petroleum ether extract of the seeds of this plant found to contain appreciable levels of secondary metabolites including phenolics, flavonoids, tannins and alkaloids that might be attributed to the good antioxidant activity of this extract. The extract exhibited broad antimicrobial activity against the tested pathogenic strains including Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella pneumonia, Candida albicans, Escherichia coli and Proteus vulgaris. 10 compounds were identified in the petroleum ether extract of Brassica juncea (L.) seeds using gas chromatography- electron ionization mass spectrometry (GC-EIMS) analysis. The identified constituents were 9,19-Cyclolanost-24-en-3-ol, acetate, (3.beta.)- Cycloartenyl acetate (34.77%), 3-Methylpentane (25.26%), Methyl cyclopentane (15.44%), octadec-9-enoic acid (oleic acid) (9.02%), Allyl isothiocyanate (3.43%), 3-Butenyl isothiocyanate (3.38%), hexanal (Caproaldehyde) (3.14%) and nonacosane (0.95%). In conclusion, Brassica juncea seeds could be used as valuable source of active constituents that possess remarkable biological activity and could be used in the field of medicine.

Carbene-based Difluoromethylation of Bisphenols: Application to the Instantaneous Tagging of Bisphenol A in Spiked Soil for Its Detection and Identification by Electron Ionization Gas Chromatography-Mass Spectrometry

The rapid and efficient difluoromethylation of a panel of eleven bisphenols (BPs) for their enhanced detection and identification by Electron-Ionization Gas Chromatography-Mass Spectrometry (EI-GC-MS) is presented. The derivatization employs the inexpensive, environmentally benign agent diethyl (bromodifluoromethyl) phosphonate (DBDFP) as a difluorocarbene-generating species that converts the BPs into bis-difluoromethylated ethers that can be detected and identified by GC-MS means. Key attributes of the protocol include its extreme rapidity (30 seconds) at ambient temperature, high specificity for BPs amidst other alcohol-containing analytes, and its biphasic nature that allows for its convenient adaptation to the analysis of BPs in organic as well as aqueous matrices. The protocol furnishes stable, novel BP ethers armed with a total of four fluorine atoms for their subsequent analysis by EI-GC-MS. Furthermore, each derivatized bisphenol exhibits unique retention times vastly different from their native counterparts leading to their unequivocal identification. The effectiveness and robustness of the developed methodology was applied to the tagging of the most famous member of this family of compounds, bisphenol-A (BPA), when spiked (at 1 μg.g−1 concentration) in the physically and compositionally complex Nebraska EPA standard soil. The method detection limit (MDL) for the bis-difluoromethylated BPA was determined to be 0.01 μg.mL−1. The bis-difluoromethylated BPA was conveniently detected on the organic layers from the biphasic, derivatized mixtures, highlighting the protocol’s practicality and utility in the rapid, qualitative detection of this endocrine disruptor during environmental analysis.

Supplemental Feeding of Laying Hens with Wood Vinegar to Decrease the Ratio of n-6 to n-3 Fatty Acids in Eggs

A balanced ratio of fatty acids n-6 to n-3 in chicken eggs is important for health and to help prevent and manage obesity and other diseases. Traditionally, fish oil or flax seed has been utilized as feed additives to decrease the ratio of n-6 to n-3(n-6:n-3) fatty acids in eggs. The hull of spina date seed(HSDS) is a common agricultural waste product in China, from which wood vinegar(HSDSWV) may be derived. This study evaluated HSDSWV as a sup-plement in hen feeds to improve the quality of eggs and decrease the ratio of fatty acids n-6:n-3. HSDSWV was obtained via carbonization, and refined. Six concentrations(nil to 0.5%) of HSDSWV were prepared and fed to 6 hen groups, respectively, for 50 d. The fatty acids of the hen’s egg yolks were analyzed by gas chromatography/electron ionization-mass spectrometry(GC/EI-MS) in the selected ion monitoring(SIM) mode. The 0.2% HSDSWV resulted in the best egg yolk quality, with a lower percentage of linoleic acid(C18:2n6) and higher percentages of cis-5,8,11,14,17-eicosapentaenoic acid(C20:5n3) and cis-4,7,10,13,16,19-docosahexaenoic acid(C22:6n3), and thus a lower n-6:n-3 ratio compared with the other HSDSWV concentrations. In addition, the eggs contained higher levels of yolk fat and egg yolk than the controls did. In conclusion, to modify the fatty acid composition of hens’ eggs and obtain a balanced ratio of n-6:n-3, 0.2% HSDSWV may be considered suitable as a dietary supplement in hens’ feed.

22 CANONICAL CORRELATION ANALYSIS OF ADHD POLYGENIC RISK AND FUNCTIONAL BRAIN CONNECTIVITY

A novel assay for the simultaneous determination of ibuprofen (IBU) and its four probable metabolites, 1-hydroxyibuprofen (1-OH IBU), 2-hydroxyibuprofen (2-OH IBU), 3-hydroxyibuprofen (3-OH IBU) and carboxyibuprofen (CBX IBU) in equine urine samples with the application of Gas Chromatography-Electron Ionization-Mass Spectrometry (GC-EI-MS) has been developed and elaborated. The new approach for sample preparation including minimizing matrix effects by the application of weak cation exchange solid-phase extraction together with strong cation exchange solid-phase extraction has been applied. The GC-EI-MS method was validated to demonstrate specificity, matrix effect, linearity, limit of detection (LOD) and quantification (LOQ), precision, trueness, carry-over and stability by using the matrix-matched quality control samples. Additionally, extraction yield was evaluated. The assay achieved the LOQ of 1.75 μg mL−1, 0.62 μg mL−1, 4.15 μg mL−1, 0.58 μg mL−1 and 4.04 μg mL−1 for IBU, 1-OH IBU, 2-OH IBU, 3-OH IBU and CBX IBU, respectively. The developed method has been successfully applied to the excretion study in horses, in which a single oral IBU dose was administered to twelve horses (mares and geldings) and equine urine samples were collected for 5 or 6 days after the drug administration. Data on the detection and determination of three IBU metabolites, 2-OH IBU, 3-OH IBU and CBX IBU in equine urine samples has been presented for the first time. The obtained results indicated the rapid excretion of IBU and its metabolites that were detectable only in the first day after the drug administration. IBU was mainly the most abundant compound detected in equine urine samples (with two exceptions in the case of samples collected from two horses, for which the highest instrumental responses were obtained for CBX IBU). The received results have indicated that two major IBU metabolites, CBX IBU and 2-OH IBU can be important markers for the IBU abuse in view of doping control in equestrian sports.

The Autoxidation of Alkenyl Succinimides—Mimics for Polyisobutenyl Succinimide Dispersants

Short chain alkenyl succinimides (ASIs) were synthesized and used as high purity chemical models to investigate the autoxidative degradation at 170 °C of polyisobutenyl succinimide dispersants (PIBSIs), a significant additive in automotive engine lubricants. Degradation products were characterized by gas chromatography–electron ionization mass spectrometry and quantified by gas chromatography-flame ionization detection. The rate of autoxidation of ASIs in a model lubricant, squalane, was also investigated. Although this is a complex molecule containing many possible sites of radical attack, all of the autoxidation products identified result from attack at the double bond or the adjacent allylic hydrogen atoms, which indicates the controlling influence of the double bond in the degradation of alkenyl succinimides, and therefore of commercial polyisobutenyl succinimide dispersants. The observed site-selective cleavage of the ASI structure, and by analogy PIBSI dispersants, would yield products that both reduce dispersancy and promote the formation of insoluble products that could have a detrimental effect on lubricant performance.

Simultaneous determination of legacy and emerging organophosphorus flame retardants and plasticizers in indoor dust using liquid and gas chromatography-tandem mass spectrometry: method development, validation, and application.

In the present study, an analytical method has been developed and validated for the simultaneous detection and quantification of 19 PFRs (14 legacy organophosphorus flame retardants (PFRs) and 5 emerging PFRs (ePFRs)) and 20 plasticizers (7 legacy plasticizers (LPs) and 13 alternative plasticizers (APs)). Sample preparation was based on the combination of previously validated analytical protocols including ultrasonic extraction and Florisil fractionation/cleanup. The analysis was performed by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for all targeted compounds, except for bis (2-ethylhexyl) phthalate (DEHP) and bis (2-ethylhexyl) terephthalate (DEHT), for which the separation of the isomers resulted in more favorable gas chromatography-electron ionization-mass spectrometry (GC-EI-MS). The new method was in-house validated by applying two levels of fortification in dust. The achieved linearity (R2) ranged between 0.993 and 0.999. Limits of detection and quantification (LODs and LOQs) ranged between 1 and 265ng/g and between 1 and 870ng/g for all analytes, respectively, except for DEHP and DEHT, for which relatively higher LODs (665 and 1100ng/g, respectively) and LOQs (2100 and 3500ng/g, respectively) were observed. Accuracy ranged between 75 and 125% for most of the targeted analytes, and repeatability was good with relative standard deviation (RSD) < 15% for most compounds. Finally, the method was applied for the determination and quantification of the targeted chemicals in house dust samples (n = 10) from the megacity of Guangzhou (China). Median values ranged from 3 to 210ng/g for PFRs, from 4 to 165ng/g for ePFRs, from 30 to 100,000ng/g for LPs, and from 6 to 34,000ng/g for APs. Main contributors to the total contamination were LPs 63% and APs 37% in total plasticizers, whereas PFRs and ePFRs contributed 90% and 10% in total flame retardants. Graphical abstract.

A gas chromatography-mass spectrometry-based whole-cell screening assay for target identification in distal cholesterol biosynthesis.

Distal cholesterol biosynthesis (CB) has recently taken center stage as a promising drug target in several diseases previously not linked to this biochemical pathway, including cardiovascular disease, cancer, multiple sclerosis and Alzheimer's disease. Most enzymes involved in this pathway are hard to isolate, warranting dedicated analytical tools for biochemical screening. We describe the use of gas chromatography-electron ionization mass spectrometry (GC-MS) in a whole-cell screening assay aimed at monitoring interactions with all enzymes of distal CB in a single experiment. Following cell culture and lipid extraction, the trimethylsilyl ethers of sterols are analyzed by GC-MS. Analytical data for 23 relevant sterols (intermediates) are provided, allowing their unambiguous identification. Sterol pattern analysis reveals the target enzyme on the basis of characteristic marker sterols, whereas quantification of 2-13C-acetate incorporation correlates with the inhibitory activity of drug candidates. The protocol can be used by both experienced scientists and newcomers to the field, allowing detection and quantification of small molecule-enzyme interactions in distal CB. The entire protocol can be carried out within two working days.

Chlorine and bromine isotope fractionations of halogenated organic compounds in fragmentation by gas chromatography-electron ionization high resolution mass spectrometry

Revealing fractionations of chlorine and bromine isotope in electron ionization mass spectrometry (EI-MS) is crucial for accurate compound-specific isotope analysis of chlorine/bromine (CSIA-Cl/Br) for halogenated organic compounds (HOCs) using gas chromatography EI-MS (GC-EI-MS). This study investigated chlorine/bromine isotope fractionation of 12 organochlorines and 5 organobromines in EI-MS using gas chromatography-high resolution mass spectrometry. The observed isotope fractionations were evaluated with relative variation of chlorine/bromine isotope ratios (Δ37Cl or Δ81Br) between precursor ions and their product ions. All the 17 HOCs exhibited significant isotope fractionations with varied modes and magnitudes depending on compound and EI energy. The magnitudes of the observed isotope fractionations were extremely large in contrast to those in in-solution dehalogenation reactions, showing the Δ37Cl and Δ81Br values within the ranges of −149.2 to 292.0‰ and −362.2 to 546.2‰, respectively. Inter-ion and intra-ion isotope fractionations counteractively influenced the measured chlorine/bromine isotope ratios of individual dehalogenation product ions, whereas only the former could affect (enhance) the measured isotope ratios of molecular ions. The magnitudes of inter-ion isotope fractionation were generally lower than those of intra-ion isotope fractionation for most HOCs (11/17). Stable EI energy and isotope-ratio calculation using complete chlorine/bromine isotopologues of individual molecular ions help to obtain CSIA-Cl/Br data with high precision and accuracy. The results of this study could facilitate exploration of chlorine and bromine isotope fractionations of HOCs during dehalogenation in such conditions as photoinduced dehalogenation, and further elucidate relevant dehalogenation pathways in light of reaction mechanisms revealed by CSIA data.

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.

Analytical validation of fecal 3-bromotyrosine concentrations in healthy dogs and dogs with chronic enteropathy.

Studies that have used serum 3-bromotyrosine (3-BrY) to investigate eosinophil activation in dogs have found elevated 3-BrY levels in clinical patients with chronic enteropathy (CE). To our knowledge, a method to measure 3-BrY concentrations in feces has not been reported. We developed and analytically validated an electron ionization gas chromatography-mass spectrometry method to measure fecal 3-BrY concentrations in dogs. The mean and maximum fecal 3-BrY concentrations in healthy dogs ( n = 40) and dogs with CE ( n = 40) over 3 consecutive days were compared. Analytical validation had a limit of blank and a limit of detection of 2.5 and 3.7 mmol/g of feces, respectively. The mean coefficients of variation for precision and reproducibility for 3-BrY were 11.2% (range: 7.5-14.2%) and 10.1% (4.8-15.2%), respectively. The ranges of observed-to-expected ratios for linearity and accuracy were 81.3-125% and 85.4-120%, respectively. The reference intervals for mean and maximum fecal 3-BrY concentrations in 40 healthy dogs were 3.7-23.0 and 3.7-37.8 mmol/g of feces. Mean and maximum fecal 3-BrY concentrations in dogs with CE were significantly higher than those of healthy dogs ( p < 0.001). Further research is warranted to determine the clinical usefulness of fecal 3-BrY concentrations in dogs with CE.