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While classical molecular biology assays can provide a measure of cellular response to chemical challenges, they rely on a single biological phenomenon to infer a broader measure of cellular metabolic response. These methods do not always afford the necessary sensitivity to answer questions of sub-cytotoxic effects, nor do they work for all cell types. Likewise, boutique assays such as cardiomyocyte beat rate may indirectly measure cellular metabolic response, but they too, are limited to measuring a specific biological phenomenon and are often limited to a single cell type. For these reasons, toxicological researchers need new approaches to determine metabolic changes across various doses in differing cell types, especially within the low-dose regime. The data collected herein demonstrate that LC-MS/MS-based untargeted metabolomics with a feature-agnostic view of the data, combined with a suite of statistical methods including an adapted environmental threshold analysis, provides a versatile, robust, and holistic approach to directly monitoring the overall cellular metabolomic response to pesticides. When employing this method in investigating two different cell types, human cardiomyocytes and neurons, this approach revealed separate sub-cytotoxic metabolomic responses at doses of 0.1 µM and 1 µM of chlorpyrifos and carbaryl. These findings suggest that this agnostic approach to untargeted metabolomics can provide a new tool for determining effective dose by metabolomics (EDm) of chemical challenges, such as pesticides, in a direct measurement of metabolomic response that is not cell type-specific or observable using traditional assays.
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Field-forward analytical technologies, such as portable mass spectrometry (MS), enable essential capabilities for real-time monitoring and point-of-care diagnostic applications. Significant and recent investments improving the features of miniaturized mass spectrometers enable various new applications outside of small molecule detection. Most notably, the addition of tandem mass spectrometry scans (MS/MS) allows the instrument to isolate and fragment ions and increase the analytical specificity by measuring unique chemical signatures for ions of interest. Notwithstanding these technological advancements, low-cost, portable systems still struggle to confidently identify clinically significant organisms of interest, such as bacteria, viruses, and proteinaceous toxins, due to the limitations in resolving power. To overcome these limitations, we developed a novel multidimensional mass fingerprinting technique that uses tandem mass spectrometry to increase the chemical specificity for low-resolution mass spectral profiles. We demonstrated the method's capabilities for differentiating four different bacteria, including attentuated strains of Yersinia pestis. This approach allowed for the accurate (>92%) identification of each organism at the strain level using de-resolved matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) data to mimic the performance characteristics of miniaturized mass spectrometers. This work demonstrates that low-resolution mass spectrometers, equipped with tandem MS acquisition modes, can accurately identify clinically relevant bacteria. These findings support the future application of these technologies for field-forward and point-of-care applications where high-performance mass spectrometers would be cost-prohibitive or otherwise impractical.
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Espectrometria de Massas em Tandem , Yersinia pestis , Yersinia pestis/isolamento & purificação , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação , Bactérias/isolamento & purificaçãoRESUMO
There is a growing need to uncover biomarkers of ionizing radiation exposure that leads to a better understanding of how exposures take place, including dose type, rate, and time since exposure. As one of the first organs to be exposed to external sources of ionizing radiation, skin is uniquely positioned in terms of model systems for radiation exposure study. The simultaneous evolution of both MS-based -omics studies, as well as in vitro 3D skin models, has created the ability to develop a far more holistic understanding of how ionizing radiation affects the many interconnected biomolecular processes that occur in human skin. However, there are a limited number of studies describing the biomolecular consequences of low-dose ionizing radiation to the skin. This review will seek to explore the current state-of-the-art technology in terms of in vitro 3D skin models, as well as track the trajectory of MS-based -omics techniques and their application to ionizing radiation research, specifically, the search for biomarkers within the low-dose range.
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Exposição à Radiação , Humanos , Modelos Biológicos , Radiação Ionizante , PeleRESUMO
Illicit drug use causes over half a million deaths worldwide every year. Drugs of abuse are commonly smuggled through customs and border checkpoints and, increasingly, through parcel delivery services. Improved methods for detection of trace drug residues from surfaces are needed. Such methods should be robust, fieldable, sensitive, and capable of detecting a wide range of drugs. In this work, commercially produced paper with a pressure-sensitive adhesive coating was utilized for the collection and analysis of trace drug residues by paper spray mass spectrometry (MS). This modified substrate was used to combine sample collection of drug residues from surfaces with rapid detection using a single paper spray ticket. The all-in-one ticket was used to probe different surfaces commonly encountered in forensic work including clothing, cardboard, glass, concrete, asphalt, and aluminum. A total of 10 drugs (acetyl fentanyl, fentanyl, clonazolam, cocaine, heroin, ketamine, methamphetamine, methylone, U-47700, and XLR-11) were evaluated and found to be detectable in the picogram range using a benchtop mass spectrometer and in the low nanogram range using a portable ion trap MS. The novel approach demonstrates a simple yet effective sampling strategy, allowing for rapid identification from difficult surfaces via paper spray mass spectrometry.
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Resíduos de Drogas , Drogas Ilícitas , Adesivos , Benzodiazepinas , Drogas Desenhadas , Drogas Ilícitas/análise , Limite de Detecção , Espectrometria de Massas , PapelRESUMO
Saliva is increasingly being targeted for metabolic studies due to its non-invasive collection methods. Tracing levels of certain metabolites within biofluids can provide indications for a myriad of physiological conditions. This study was performed on a panel of eight analytes found in saliva that have shown associations with physiological conditions of human performance, such as stress, inflammation, and circadian rhythm. This dual polarity liquid chromatography tandem mass spectrometric (LCMS/MS) method was developed to accommodate a diverse group of analytes including steroids, alkaloids, and neurotransmitters. Samples collected during field exercises from soldiers were compared to those of civilians and baseline levels of each of these compounds was determined in saliva. Although most analytes showed no significant differences between the two populations, relative cortisol levels were higher for soldiers than for civilians. This developed dual polarity LCMS/MS method can be applied to very diverse groups of salivary analytes simultaneously.
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Cromatografia Líquida , Testes de Química Clínica/métodos , Dopagem Esportivo/prevenção & controle , Substâncias para Melhoria do Desempenho/análise , Saliva/química , Espectrometria de Massas em Tandem , Alcaloides/análise , Humanos , Neurotransmissores/análise , Substâncias para Melhoria do Desempenho/metabolismo , Esteroides/análiseRESUMO
Despite the recent epidemic of fentanyl abuse, there are few validated assays capable of rapidly detecting these compounds. In order to improve the ability to detect carfentanil at physiologically relevant concentrations, we developed a systems biology approach to discover host-based markers which are specifically amplified upon exposure in a rabbit model. For this work, two "omics" pipelines utilizing mass spectrometry were developed and leveraged. First, a proteomics pipeline was developed to interrogate the blood plasma for protein-based biomarkers. Due to the incredible dynamic range of the plasma protein content, a multi-dimensional fractionation technique was used to partition and more accurately investigate the circulating plasma proteome. Isobaric tandem mass tags were integrated into the workflow to make quantitative assessments across all animals for an extended time course post-exposure. In addition to the proteomics efforts, blood plasma was also processed through an untargeted metabolomics pipeline. This approach allows for the identification of >800 small molecule features. By processing and analyzing data sets in parallel, we were able to identify a unique fingerprint of protein and metabolite perturbations that manifest following exposure to carfentanil.
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Analgésicos Opioides/sangue , Exposição Ambiental/análise , Fentanila/análogos & derivados , Exposição por Inalação/análise , Metabolômica/métodos , Proteômica/métodos , Animais , Biomarcadores/sangue , Proteínas Sanguíneas/análise , Cromatografia de Fase Reversa , Fentanila/sangue , Masculino , Espectrometria de Massas , Metaboloma/efeitos dos fármacos , Metabolômica/instrumentação , Proteômica/instrumentação , CoelhosRESUMO
Currently, all assays measuring acetylcholinesterase (AChE) activity following a suspected nerve agent exposure leverage methodologies that fail to identify the agent. This limits the overall effectiveness and ability to administer proper countermeasures. As such, there is an urgent need to identify novel, rapid, and more comprehensive approaches to establish AChE activity, including identification of the toxicant. Paper spray mass spectrometry was used to monitor the activity of acetylcholinesterase, both in-solution and on modified hydrophobic paper surface. Hydrophobic paper surfaces were prepared using vaporized trichloro(3,3,3-trifluoropropyl)silane. In both approaches, mixtures of diluted human whole blood with and without VX were mixed with a non-endogenous AChE specific substrate, 1,1-dimethyl-4-acetylthiomethylpiperidinium (MATP+). Formation of the cleaved MATP+ product was monitored over time and compared to MATP+ to determine relative AChE activity. This on-substrate assay was effective at determining AChE activity and identifying the toxicant; however, determination of AChE activity in-solution proceeded at a slower rate. The on-substrate assay serves as a pioneering example of an enzymatic reaction occurring on the surface of a paper spray ionization ticket. This work broadens the range of applications relating to paper spray ionization-based clinical diagnostic assays. Graphical Abstract á .
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Acetilcolinesterase/sangue , Acetilcolinesterase/metabolismo , Ensaios Enzimáticos/métodos , Espectrometria de Massas/métodos , Humanos , Papel , PiperidinasRESUMO
RATIONALE: The analysis of chemical warfare agents (CWAs) from ambient atmosphere presents an analytical challenge due to their ease of degradation and volatility. Herein is described a method for derivatizing CWAs directly onto a paper spray substrate prior to analysis. This derivatization allows for much longer times of analysis without sample degradation and with little to no sample preparation. METHODS: Derivatization was performed using 2-[(dimethylamino)methyl] phenol both in-vial and directly on paper spray cartridges. Solution studies were carried out over time and samples were analyzed via liquid chromatography/tandem mass spectrometry (LC/MS/MS) operated in positive ion mode. Paper spray substrates impregnated with the derivatizing agent prior to CWA vapor capture were also analyzed over time using a mass spectrometer operated in positive ion mode. RESULTS: Use of 2-[(dimethylamino)methyl] phenol as a paper spray substrate dopant enables derivatization of G-series compounds into lower volatility complexes. The reaction occurs in solution and in the vapor phase. This new technique effectively traps and captures G-series agents for analysis while extending the time for which the compound remains absorbed. The complex is highly suitable for direct analysis via paper spray mass spectrometry. CONCLUSIONS: Derivatization of paper spray substrates was shown to greatly increase the time for analysis of CWAs. This technique, combined with the vapor phase capture stage outlined previously, allows for rapid, quantitative CWA detection by paper spray ionization with little or no sample preparation.
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Substâncias para a Guerra Química/química , Espectrometria de Massas em Tandem/métodos , Compostos Orgânicos Voláteis/química , Cromatografia Líquida/métodos , PapelRESUMO
Since their inception, mass spectrometers have played a pivotal role in the direction and application of synthetic chemical research. The ability to develop new instrumentation to solve current analytical challenges in this area has always been at the heart of mass spectrometry, although progress has been slow at times. Herein, we briefly review the history of how mass spectrometry has been used to approach challenges in organic chemistry, how new developments in portable instrumentation and ambient ionization have been used to open novel areas of research, and how current techniques have the ability to expand on our knowledge of synthetic mechanisms and kinetics. Lastly, we discuss the relative paucity of work done in recent years to embrace the concept of improving benchtop synthetic chemistry with mass spectrometry, the disconnect between applications and fundamentals within these studies, and what hurdles still need to be overcome. Graphical Abstract.
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Impurity profiling has been used as a useful tool for analyzing nearly every drug class currently known on the illicit market. Impurities present within seized samples have the potential to determine source of origin, route of synthesis used, as well as provide a useful clue into the potential reaction mechanisms that are present for each synthetic procedure. Perhaps the most well studied of these impurity profiles exists for methamphetamine, including information to more than one route of synthesis. Within the present study, a complete synthesis of methamphetamine was performed, including a reductive amination of phenylpropanone (P2P) using methylamine hydrochloride and sodium triacetoxyborohydride (STAB) rather than the conventional aluminum mercury amalgam commonly found in the literature. During the analysis of the final product from this reaction, a major impurity within the reaction, bis(1-phenylpropan-2-yl)amine (m/z 253), was detected by GC-MS as well as direct-infusion ESI-MS. This impurity has been previously reported as a Leuckart route-specific impurity. Its detection within the reductive amination of P2P points towards the use of impure methylamine hydrochloride containing some traces of acid, and provides further insight into the reductive amination of P2P. In both the Leuckart reaction and this reductive amination via STAB, the presence of acid and ammonia leads to this impurity.
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Contaminação de Medicamentos , Drogas Ilícitas/síntese química , Metanfetamina/síntese química , Aminação , Cromatografia Gasosa-Espectrometria de Massas , OxirreduçãoRESUMO
We present an analysis of ambient benzene, toluene, and xylene isomers in the Eagle Ford shale region of southern Texas. In situ air quality measurements using membrane inlet mobile mass spectrometry revealed ambient benzene and toluene concentrations as high as 1000 and 5000 parts-per-billion, respectively, originating from specific sub-processes on unconventional oil and gas well pad sites. The detection of highly variant contamination events attributable to natural gas flaring units, condensate tanks, compressor units, and hydrogen sulfide scavengers indicates that mechanical inefficiencies, and not necessarily the inherent nature of the extraction process as a whole, result in the release of these compounds into the environment. This awareness of ongoing contamination events contributes to an enhanced knowledge of ambient volatile organic compounds on a regional scale. While these reconnaissance measurements on their own do not fully characterize the fluctuations of ambient BTEX concentrations that likely exist in the atmosphere of the Eagle Ford Shale region, they do suggest that contamination events from unconventional oil and gas development can be monitored, controlled, and reduced.
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Poluentes Atmosféricos/análise , Benzeno/análise , Monitoramento Ambiental/métodos , Indústria de Petróleo e Gás , Tolueno/análise , Xilenos/análise , Poluição do Ar , TexasRESUMO
The ability to detect atmospheric effluent from clandestine methamphetamine manufacture is a useful tool for law enforcement. A membrane inlet mass spectrometer is mounted onto an all-electric drive capable hybrid vehicle that samples the atmosphere while in motion. Attributing a latitude and longitude to each spectrum collected, unique chemical fingerprints from clandestine manufacture are then mapped. This location-based mass spectrum data provides a localization to an area of interest. The synthesis of methamphetamine precursors was performed, and the impurities from such reactions were observed. A mock manufacture was setup, and the impurities were introduced into the atmosphere via heating. The detection of products and impurities using this mobile platform has shown the effectiveness of locating and localizing the manufacture of methamphetamine.