Application of Ion Mobility Spectrometry-Mass Spectrometry for Compositional Characterization and Fingerprinting of a Library of Diverse Crude Oil Samples.

Exposure characterization of crude oils, especially in time-sensitive circumstances such as spills and disasters, is a well-known analytical chemistry challenge. Gas chromatography-mass spectrometry is commonly used for "fingerprinting" and origin tracing in oil spills; however, this method is both time-consuming and lacks the resolving power to separate co-eluting compounds. Recent advances in methodologies to analyze petroleum substances using high-resolution analytical techniques have demonstrated both improved resolving power and higher throughput. One such method, ion mobility spectrometry-mass spectrometry (IMS-MS), is especially promising because it is both rapid and high-throughput, with the ability to discern among highly homologous hydrocarbon molecules. Previous applications of IMS-MS to crude oil analyses included a limited number of samples and did not provide detailed characterization of chemical constituents. We analyzed a diverse library of 195 crude oil samples using IMS-MS and applied a computational workflow to assign molecular formulas to individual features. The oils were from 12 groups based on geographical and geological origins: non-US (1 group), US onshore (3), and US Gulf of Mexico offshore (8). We hypothesized that information acquired through IMS-MS data would provide a more confident grouping and yield additional fingerprint information. Chemical composition data from IMS-MS was used for unsupervised hierarchical clustering, as well as machine learning-based supervised analysis to predict geographic and source rock categories for each sample; the latter also yielded several novel prospective biomarkers for fingerprinting of crude oils. We found that IMS-MS data have complementary advantages for fingerprinting and characterization of diverse crude oils and that proposed polycyclic aromatic hydrocarbon biomarkers can be used for rapid exposure characterization. Environ Toxicol Chem 2023;42:2336-2349. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Integrative Chemical-Biological Grouping of Complex High Production Volume Substances from Lower Olefin Manufacturing Streams.

Human cell-based test methods can be used to evaluate potential hazards of mixtures and products of petroleum refining ("unknown or variable composition, complex reaction products, or biological materials" substances, UVCBs). Analyses of bioactivity and detailed chemical characterization of petroleum UVCBs were used separately for grouping these substances; a combination of the approaches has not been undertaken. Therefore, we used a case example of representative high production volume categories of petroleum UVCBs, 25 lower olefin substances from low benzene naphtha and resin oils categories, to determine whether existing manufacturing-based category grouping can be supported. We collected two types of data: nontarget ion mobility spectrometry-mass spectrometry of both neat substances and their organic extracts and in vitro bioactivity of the organic extracts in five human cell types: umbilical vein endothelial cells and induced pluripotent stem cell-derived hepatocytes, endothelial cells, neurons, and cardiomyocytes. We found that while similarity in composition and bioactivity can be observed for some substances, existing categories are largely heterogeneous. Strong relationships between composition and bioactivity were observed, and individual constituents that determine these associations were identified. Overall, this study showed a promising approach that combines chemical composition and bioactivity data to better characterize the variability within manufacturing categories of petroleum UVCBs.

Connexin 40-Mediated Regulation of Systemic Circulation and Arterial Blood Pressure.

Vascular system is a complex network in which different cell types and vascular segments must work in concert to regulate blood flow distribution and arterial blood pressure. Although paracrine/autocrine signaling is involved in the regulation of vasomotor tone, direct intercellular communication via gap junctions plays a central role in the control and coordination of vascular function in the microvascular network. Gap junctions are made up by connexin (Cx) proteins, and among the four Cxs expressed in the cardiovascular system (Cx37, Cx40, Cx43, and Cx45), Cx40 has emerged as a critical signaling pathway in the vessel wall. This Cx is predominantly found in the endothelium, but it is involved in the development of the cardiovascular system and in the coordination of endothelial and smooth muscle cell function along the length of the vessels. In addition, Cx40 participates in the control of vasomotor tone through the transmission of electrical signals from the endothelium to the underlying smooth muscle and in the regulation of arterial blood pressure by renin-angiotensin system in afferent arterioles. In this review, we discuss the participation of Cx40-formed channels in the development of cardiovascular system, control and coordination of vascular function, and regulation of arterial blood pressure.

Analytical chemistry solutions to hazard evaluation of petroleum refining products.

Products of petroleum refining are substances that are both complex and variable. These substances are produced and distributed in high volumes; therefore, they are heavily scrutinized in terms of their potential hazards and risks. Because of inherent compositional complexity and variability, unique challenges exist in terms of their registration and evaluation. Continued dialogue between the industry and the decision-makers has revolved around the most appropriate approach to fill data gaps and ensure safe use of these substances. One of the challenging topics has been the extent of chemical compositional characterization of products of petroleum refining that may be necessary for substance identification and hazard evaluation. There are several novel analytical methods that can be used for comprehensive characterization of petroleum substances and identification of most abundant constituents. However, translation of the advances in analytical chemistry to regulatory decision-making has not been as evident. Therefore, the goal of this review is to bridge the divide between the science of chemical characterization of petroleum and the needs and expectations of the decision-makers. Collectively, mutual appreciation of the regulatory guidance and the realities of what information these new methods can deliver should facilitate the path forward in ensuring safety of the products of petroleum refining.

Characterization of Compositional Variability in Petroleum Substances.

In the process of registration of substances of Unknown or Variable Composition, Complex Reaction Products or Biological Materials (UVCBs), information sufficient to enable substance identification must be provided. Substance identification for UVCBs formed through petroleum refining is particularly challenging due to their chemical complexity, as well as variability in refining process conditions and composition of the feedstocks. This study aimed to characterize compositional variability of petroleum UVCBs both within and across product categories. We utilized ion mobility spectrometry (IMS)-MS as a technique to evaluate detailed chemical composition of independent production cycle-derived samples of 6 petroleum products from 3 manufacturing categories (heavy aromatic, hydrotreated light paraffinic, and hydrotreated heavy paraffinic). Atmospheric pressure photoionization and drift tube IMS-MS were used to identify structurally related compounds and quantified between- and within-product variability. In addition, we determined both individual molecules and hydrocarbon blocks that were most variable in samples from different production cycles. We found that detailed chemical compositional data on petroleum UVCBs obtained from IMS-MS can provide the information necessary for hazard and risk characterization in terms of quantifying the variability of the products in a manufacturing category, as well as in subsequent production cycles of the same product.

Analysis of per- and polyfluoroalkyl substances in Houston Ship Channel and Galveston Bay following a large-scale industrial fire using ion-mobility-spectrometry-mass spectrometry.

Per- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants of concern because of their ubiquitous presence in surface and ground water; analytical methods that can be used for rapid comprehensive exposure assessment and fingerprinting of PFAS are needed. Following the fires at the Intercontinental Terminals Company (ITC) in Deer Park, TX in 2019, large quantities of PFAS-containing firefighting foams were deployed. The release of these substances into the Houston Ship Channel/Galveston Bay (HSC/GB) prompted concerns over the extent and level of PFAS contamination. A targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based study of temporal and spatial patterns of PFAS associated with this incident revealed presence of 7 species; their levels gradually decreased over a 6-month period. Because the targeted LC-MS/MS analysis was focused on about 30 PFAS molecules, it may have missed other PFAS compounds present in firefighting foams. Therefore, we utilized untargeted LC-ion mobility spectrometry-mass spectrometry (LC-IMS-MS)-based analytical approach for a more comprehensive characterization of PFAS in these water samples. We analyzed 31 samples from 9 sites in the HSC/GB that were collected over 5 months after the incident. Our data showed that additional 19 PFAS were detected in surface water of HSC/GB, most of them decreased gradually after the incident. PFAS features detected by LC-MS/MS correlated well in abundance with LC-IMS-MS data; however, LC-IMS-MS identified a number of additional PFAS, many known to be components of firefighting foams. These findings therefore illustrate that untargeted LC-IMS-MS improved our understanding of PFAS presence in complex environmental samples.

Dosing Methods to Enable Cell-Based In Vitro Testing of Complex Substances: A Case Study with a PAH Mixture.

Cell-based testing of multi-constituent substances and mixtures for their potential adverse health effects is difficult due to their complex composition and physical-chemical characteristics. Various extraction methods are typically used to enable studies in vitro; however, a limited number of solvents are biocompatible with in vitro studies and the extracts may not fully represent the original test article's composition. While the methods for dosing with "difficult-to-test" substances in aquatic toxicity studies are well defined and widely used, they are largely unsuited for small-volume (100 microliters or less) in vitro studies with mammalian cells. Therefore, we aimed to evaluate suitability of various scaled-down dosing methods for high-throughput in vitro testing by using a mixture of polycyclic aromatic hydrocarbons (PAH). Specifically, we compared passive dosing via silicone micro-O-rings, cell culture media-accommodated fraction, and traditional solvent (dimethyl sulfoxide) extraction procedures. Gas chromatography-tandem mass spectrometry (GC-MS/MS) was used to evaluate kinetics of PAH absorption to micro-O-rings, as well as recovery of PAH and the extent of protein binding in cell culture media with and without cells for each dosing method. Bioavailability of the mixture from different dosing methods was also evaluated by characterizing in vitro cytotoxicity of the PAH mixture using EA.hy926 and HepG2 human cell lines. Of the tested dosing methods, media accommodated fraction (MAF) was determined to be the most appropriate method for cell-based studies of PAH-containing complex substances and mixtures. This conclusion is based on the observation that the highest fraction of the starting materials can be delivered using media accommodated fraction approach into cell culture media and thus enable concentration-response in vitro testing.

Practical One-Pot Multistep Synthesis of 2H-1,3-Benzoxazines Using Copper, Hydrogen Peroxide and Triethylamine.

In this article, we describe simple one-pot syntheses of 2H-1,3-benzoxazines from ketones utilizing an imino-pyridine directing group (R1R2-C=N-CH2-Pyr), which promotes a Cu-directed sp2 hydroxylation using H2O2 as oxidant and followed by an oxidative intramolecular C-O bond formation upon addition of NEt3. This synthetic protocol is utilized in the gram scale synthesis of the 2H-1,3-benzoxazine derived from benzophenone. Mechanistic studies reveal that the cyclization occurs via deprotonation of the benzylic position of the directing group to produce a 2-azallyl anion intermediate, which is oxidized to the corresponding 2-azaallyl radical before the C-O bond formation event. Understanding of the cyclization mechanism also allowed us to develop reaction conditions that utilize catalytic amounts of Cu.

Cu-promoted intramolecular hydroxylation of CH bonds using directing groups with varying denticity.

In this research article, we describe the Cu-promoted intramolecular hydroxylation of sp2 and sp3 CH bonds using directing groups with varying denticity (bi-, tri- and tetradentate) and natural oxidants (O2 and H2O2). We found that bidentate directing groups, in combination with Cu and H2O2, led to high hydroxylation yields. On the other hand, tetradentate directing groups did not form the hydroxylation products. Our mechanistic investigations suggest that bidentate directing groups allow for generating reactive mononuclear copper(II) hydroperoxide intermediates while tetradentate systems form dinuclear Cu2O2 species that do not oxidize CH bonds. Our findings might shed light on the reaction mechanism(s) by which Cu-dependent metalloenzymes such as particulate methane monooxygenase or lytic polysaccharide monooxygenase oxidize strong CH bonds.

Data Processing Workflow to Identify Structurally Related Compounds in Petroleum Substances Using Ion Mobility Spectrometry-Mass Spectrometry.

Ion mobility spectrometry coupled with mass spectrometry (IMS-MS) is a post-ionization separation technique that can be used for rapid multidimensional analyses of complex samples. IMS-MS offers untargeted analysis, including ion-specific conformational data derived as collisional cross section (CCS) values. Here, we combine nitrogen gas drift tube CCS (DTCCSN2) and Kendrick mass defect (KMD) analyses based on CH2 and H functional units to enable compositional analyses of petroleum substances. First, polycyclic aromatic compound standards were analyzed by IMS-MS to demonstrate how CCS assists the identification of isomeric species in homologous series. Next, we used case studies of a gasoline standard previously characterized for paraffin, isoparaffin, aromatic, naphthene, and olefinic (PIANO) compounds, and a crude oil sample to demonstrate the application of the KMD analyses and CCS filtering. Finally, we propose a workflow that enables confident molecular formula assignment to the IMS-MS-derived features in petroleum samples. Collectively, this work demonstrates how rapid untargeted IMS-MS analysis and the proposed data processing workflow can be used to provide confident compositional characterization of hydrocarbon-containing substances.

Directed Hydroxylation of sp2 and sp3 C-H Bonds Using Stoichiometric Amounts of Cu and H2O2.

The use of copper for C-H bond functionalization, compared to other metals, is relatively unexplored. Herein, we report a synthetic protocol for the regioselective hydroxylation of sp2 and sp3 C-H bonds using a directing group, stoichiometric amounts of Cu and H2O2. A wide array of aromatic ketones and aldehydes are oxidized in the carbonyl γ-position with remarkable yields. We also expanded this methodology to hydroxylate the ß-position of alkylic ketones. Spectroscopic characterization, kinetics, and density functional theory calculations point toward the involvement of a mononuclear LCuII(OOH) species, which oxidizes the aromatic sp2 C-H bonds via a concerted heterolytic O-O bond cleavage with concomitant electrophilic attack on the arene system.

PGC-1α-Dependent Mitochondrial Adaptation Is Necessary to Sustain IL-2-Induced Activities in Human NK Cells.

Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that, after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass and the mitochondrial membrane potential increased in a PGC-1α-dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γ from NK cells with LAK activity was also partially dependent on PGC-1α expression. These results indicate that PGC-1α plays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2.