Formation of Diacetyl and Other α-Dicarbonyl Compounds during the Generation of E-Vapor Product Aerosols.

Exposure to diacetyl (DA) has been linked to the respiratory condition bronchiolitis obliterans. Previous research has demonstrated that DA and other α-dicarbonyl compounds can be detected in both the e-liquids and aerosols of e-vapor products (EVPs). While some EVP manufacturers may add these compounds as flavor ingredients, the primary objective of this work was to determine the potential for the formation of α-dicarbonyl compounds during the generation of aerosols from EVPs where no DA or other α-dicarbonyl compounds are added to the e-liquid. A novel ultraperformance liquid chromatography-mass spectrometry-based analytical method for the determination of DA, acetyl propionyl, glyoxal, and methylglyoxal was developed and validated. Next, eight commercially available cig-a-like-type EVPs were evaluated for α-dicarbonyl formation. Increased levels of α-dicarbonyls were observed in the aerosols of all evaluated EVPs compared to their respective e-liquids. Mechanistic studies were conducted using a model microwave reaction system to identify key reaction precursors for DA generated from propylene glycol (PG) and carbon-13-labeled glycerin (GLY). These studies, along with the corresponding retrosynthetic analysis, resulted in the proposed formation pathway where hydroxyacetone is generated from PG and/or GLY. Hydroxyacetone then participates in an aldol condensation with formaldehyde where formaldehyde can also be generated from PG and/or GLY; the resultant product then dehydrates to form DA. This proposed pathway was further investigated through in situ synthetic organic experiments within the model microwave reaction system. This work establishes that DA is formed in the aerosol generation process of the EVPs tested though at levels below toxicological concern.

An evaluation of electronic cigarette formulations and aerosols for harmful and potentially harmful constituents (HPHCs) typically derived from combustion.

U.S. FDA draft guidance recommends reporting quantities of designated harmful and potentially harmful constituents (HPHCs) in e-cigarette e-liquids and aerosols. The HPHC list comprises potential matrix-related compounds, flavors, nicotine, tobacco-related impurities, leachables, thermal degradation products, and combustion-related compounds. E-cigarettes contain trace levels of many of these constituents due to tobacco-derived nicotine and thermal degradation. However, combustion-related HPHCs are not likely to be found due to the relatively low operating temperatures of most e-cigarettes. The purpose of this work was to use highly sensitive, selective, and validated analytical methods to determine if these combustion-related HPHCs (three aromatic amines, five volatile organic compounds, and the polycyclic aromatic hydrocarbon benzo[a]pyrene) are detectable in commercial refill e-liquids, reference e-cigarette e-liquids, and aerosols generated from rechargeable e-cigarettes with disposable cartridges (often referred to as "cig-a-likes"). In addition, the transfer efficiency of these constituents from e-liquid to aerosol was evaluated when these HPHCs were added to the e-liquids prior to aerosol formation. This work demonstrates that combustion-related HPHCs are not present at measurable levels in the commercial and reference e-liquids or e-cigarette aerosols tested. Additionally, when combustion-related HPHCs are added to the e-liquids, they transfer to the aerosol with transfer efficiencies ranging from 49% to 99%.

Gas Chromatography-Mass Spectrometry Method to Quantify Benzo[a]Pyrene in Tobacco Products.

The USA Food and Drug Administration (FDA) established benzo[a]pyrene (B[a]P) as a harmful and potentially harmful constituent (HPHC) found in tobacco products. Tobacco manufacturers are required to report HPHC quantities to the FDA; however, there is currently no standardized method for determination of B[a]P in smokeless tobacco products (STPs). This work details a sensitive, selective and rapid method for the determination of B[a]P in STPs, cigarette filler and tobacco. Tobacco is extracted using methanol followed by solid-phase extraction and concentration prior to analysis by gas chromatography/mass spectrometry in the selected ion monitoring mode. Cooperation Centre for Scientific Research Relative to Tobacco reference products and 3R4F Kentucky reference cigarette filler were used for method validation. All method validation requirements were met including linearity, accuracy, precision, robustness, limit of detection (LOD) and limit of quantitation (LOQ), and stability. Calibration range of 0.5-125 ng mL-1 was achieved with the coefficient of determination (R2) greater than 0.995. The method LOQ and LOD were 0.729 and 0.216 ng/g, respectively. Using standardized methods for the measurement of HPHCs in tobacco products will reduce variability and ensure accurate data for regulatory reporting.

Method for the Determination of Carbonyl Compounds in E-Cigarette Aerosols.

Low levels of thermal degradation products such as carbonyls (formaldehyde, acetaldehyde, acrolein, crotonaldehyde) have been reported in e-cigarette aerosols. The collection and analysis of e-cigarette aerosol carbonyls are often adapted from methods developed for tobacco cigarette smoke. These methodologies are often not sensitive enough to detect low carbonyl levels in e-cigarette aerosols. One objective of this work was to develop and validate a rapid, selective and sensitive ultra-performance liquid chromatography with mass spectrometry method optimized for analysis of carbonyls in e-cigarette aerosols. Aerosols were trapped in 20-puff collections, 4-s durations, 55-mL volumes, 30-s intervals, square wave puff profiles. Collection apparatus involved a linear smoking machine with Cambridge filter pad followed by a glass impinger containing acidified 2,4-dinitrophenylhydrazine. This method showed limits of quantitation and detection of 0.016 and 0.003 µg puff-1, respectively, and run time of 4 min. Six e-cigarettes were evaluated (five devices each). All contained measurable levels of carbonyls. Levels were mostly well below those in conventional cigarettes. However, for some e-cigarettes, formaldehyde levels were above those for tobacco cigarettes (highest at 14.1 µg puff-1). Temperatures related to carbonyl yields in e-cigarette aerosols were explored to better understand carbonyl formation: formation of formaldehyde is low at temperatures below 350°C.

Characterization of potential impurities and degradation products in electronic cigarette formulations and aerosols.

E-cigarettes are gaining popularity in the U.S. as well as in other global markets. Currently, limited published analytical data characterizing e-cigarette formulations (e-liquids) and aerosols exist. While FDA has not published a harmful and potentially harmful constituent (HPHC) list for e-cigarettes, the HPHC list for currently regulated tobacco products may be useful to analytically characterize e-cigarette aerosols. For example, most e-cigarette formulations contain propylene glycol and glycerin, which may produce aldehydes when heated. In addition, nicotine-related chemicals have been previously reported as potential e-cigarette formulation impurities. This study determined e-liquid formulation impurities and potentially harmful chemicals in aerosols of select commercial MarkTen(®) e-cigarettes manufactured by NuMark LLC. The potential hazard of the identified formulation impurities and aerosol chemicals was also estimated. E-cigarettes were machine puffed (4-s duration, 55-mL volume, 30-s intervals) to battery exhaustion to maximize aerosol collection. Aerosols analyzed for carbonyls were collected in 20-puff increments to account for analyte instability. Tobacco specific nitrosamines were measured at levels observed in pharmaceutical grade nicotine. Nicotine-related impurities in the e-cigarette formulations were below the identification and qualification thresholds proposed in ICH Guideline Q3B(R2). Levels of potentially harmful chemicals detected in the aerosols were determined to be below published occupational exposure limits.

Proteomic biomarkers in plasma that differentiate rapid and slow decline in lung function in adult cigarette smokers with chronic obstructive pulmonary disease (COPD).

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of morbidity and mortality in the United States and cigarette smoking is a primary determinant of the disease. COPD is characterized by chronic airflow limitation as measured by the forced expiratory volume in one second (FEV(1)). In this study, the plasma proteomes of 38 middle-aged or older adult smokers with mild to moderate COPD, with FEV(1) decline characterized as either rapid (RPD, n = 20) or slow or absent (SLW, n = 18), were interrogated using a comprehensive high-throughput proteomic approach, the accurate mass and time (AMT) tag technology. This technology is based upon a putative mass and time tag database (PMT), high-resolution LC separations and high mass accuracy measurements using FT-ICR MS with a 9.4-T magnetic field. The peptide and protein data were analyzed using three statistical approaches to address ambiguities related to the high proportion of missing data inherent to proteomic analysis. The RPD and SLW groups were differentiated by 55 peptides which mapped to 33 unique proteins. Twelve of the proteins have known roles in the complement or coagulation cascade and, despite an inability to adjust for some factors known to affect lung function decline, suggest potential mechanistic biomarkers associated with the rate of lung function decline in COPD. Whether these proteins are the cause or result of accelerated decline will require further research.

High-resolution mass spectrometry proteomics for the identification of candidate plasma protein biomarkers for chronic obstructive pulmonary disease.

Although cigarette smoking is recognized as the most important cause of chronic obstructive pulmonary disease (COPD), the pathophysiological mechanisms underlying the lung function decline are not well understood. Using off-line strong cation exchange fractionation with RP-LC-ESI-MS/MS and robust database searching, 1758 tryptic peptides were identified in plasma samples from cigarette smokers. Using two statistical approaches, 30 peptides were identified to be associated with the annualized rate of lung function decline over 5 years among smokers with COPD characterized as having rapid (n = 18) or slow (n = 18) decline and 18 smokers without COPD. The identified peptides belong to proteins that are involved in the complement or coagulation systems or have antiprotease or metabolic functions. This research demonstrates the utility of proteomic profiling to improve the understanding of molecular mechanisms involved in cigarette smoking-related COPD by identifying plasma proteins that correlate with decline in lung function.

Cigarette smoke extract induced protein phosphorylation changes in human microvascular endothelial cells in vitro.

Phosphorylation is the most widely studied posttranslational modification (PTM) and is an important regulatory mechanism used during cellular responses to external stimuli. The kinases and phosphatases that regulate protein phosphorylation are known to be affected in many human diseases. Cigarette smoking causes cardiovascular disease (CVD). Endothelial cells play a pivotal role in CVD initiation and development; however, there have been limited investigations of the specific signaling cascades and protein phosphorylations activated by cigarette smoke in endothelial cells. The purpose of this research was to better understand the differential protein phosphorylation in endothelial cells stimulated with extracts of cigarette smoke total particulate matter (CS-TPM) in vitro. Human microvascular endothelial cells were exposed in vitro to CS-TPM at concentrations that were shown to cause endothelial cell dysfunction. The phosphorylated proteins were isolated using phosphoprotein-specific chromatography, followed by enzymatic digestion and nano-flow capillary liquid chromatography (ncap-LC) coupled to high resolution mass spectrometry. This study putatively identified 94 proteins in human microvascular endothelial cells that were differentially bound to a phosphoprotein-specific chromatography column following exposure to CS-TPM suggesting differential phosphorylation. Pathway analysis has also been conducted and confirmations of several observations have been made using immunoaffinity-based techniques (e.g., Western blotting).

Carbon-centered radicals in cigarette smoke: acyl and alkylaminocarbonyl radicals.

The widely accepted mechanism of formation for carbon-centered radicals in the gas-phase cigarette smoke involves reactions of NO(2) and alkadienes. However, specific examples of such radicals have never been isolated from fresh cigarette smoke or their structure determined. We have identified two previously unrecognized classes of carbon-centered radicals, alkylaminocarbonyl and acyl radicals, that are unrelated to radicals that form by NO(x) chemistry. The combined abundance of these mainstream smoke radicals is significantly higher than the alkyl radicals previously quantified by electron paramagnetic resonance (EPR) solution spin-trapping methods. The new radicals were trapped directly from smoke with either 3-amino-proxyl (3AP) or 3-cyano-proxyl radical on a solid support and identified by combination of chemical synthesis, deuterium labeling, high-resolution mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and ab initio quantum mechanical calculations. 3AP-R adducts were quantified both by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and by high-performance liquid chromatography with fluorescence detection (HPLC/FLD). Seven acyl and 11 alkylaminocarbonyl radicals were identified in the whole smoke of cigarettes made from single tobacco varieties and blended tobacco research cigarettes. The overall yield of these radicals was measured to be 168-245 nmol/cigarette from machine-smoked cigarettes under Federal Trade Commission (FTC) conditions. The yield was significantly reduced when the gas-phase smoke was separated from whole smoke by filtration through a 0.1 microm Cambridge filter pad or upon aging whole smoke in an inert tube.

Phosphine polymerization by nitric oxide: experimental characterization and theoretical predictions of mechanism.

A yellow solid material [P(x)H(y)] has been obtained in the reaction of phosphine (PH(3)) and nitric oxide (NO) at room temperature and characterized by thermogravimetric analysis mass spectrometry (TGA-MS) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. In this work using complete basis set (CBS-QB3) methods a plausible mechanism has been investigated for phosphine polymerization in the presence of nitric oxide (NO). Theoretical explorations with the ab initio method suggest (a) instead of the monomer the nitric oxide dimer acts as an initial oxidant, (b) the resulting phosphine oxides (H(3)P=O <--> H(3)P(+)O(-)) in the gas phase draw each other via strong dipolar interactions between the P-O groups, and (c) consequently an autocatalyzed polymerization occurs among the phosphine oxides, forming P-P chemical bonds and losing water. The possible structures of polyhydride phosphorus polymer were discussed. In the calculations a series of cluster models was computed to simulate polymerization.

Identification of in vitro differential cell secretions due to cigarette smoke condensate exposure using nanoflow capillary liquid chromatography and high-resolution mass spectrometry.

Secreted proteins, the secretome, can be isolated from biological fluids (e.g., blood) and are often responsible for the regulation of biological processes such as cell signaling, growth, and apoptosis. The identification of secreted proteins can lead to an understanding of disease mechanisms and they can serve as early candidate biomarkers of disease and exposure. However, it is time-consuming and costly to conduct in vivo interrogations of the human secretome. The purpose of this article is to provide a detailed description of a rapid in vitro technique for the analysis of differential protein secretion due to exposure to smoking-machine-generated cigarette smoke (CS) condensate (total particulate matter, TPM). Endothelial cells were exposed to CS-TPM, the supernatant was collected, and the secretome was elucidated by nano liquid chromatography coupled with high-resolution mass spectrometry. A total of 1,677 unique peptides were identified in the cell culture supernatants. Several proteins were differentially expressed following CS-TPM exposure that relate to several biological processes, such as metabolism, development, communication, response to stimulus, and response to stress.

Characterization of the mouse bronchoalveolar lavage proteome by micro-capillary LC-FTICR mass spectrometry.

Bronchoalveolar lavage fluid (BALF) contains proteins derived from various pulmonary cell types, secretions and blood. As the characterization of the BALF proteome will be instrumental in establishing potential biomarkers of pathophysiology in the lungs, the objective of this study was to contribute to the comprehensive collection of Mus musculus BALF proteins using high resolution and highly sensitive micro-capillary liquid chromatography (microLC) combined with state-of-the-art high resolution mass spectrometry (MS). BALF was collected from ICR and C57BL/6 male mice exposed to nose-only inhalation to either air or cigarette smoke. The tandem mass spectra were analyzed by SEQUEST for peptide identifications with the subsequent application of accurate mass and time tags resulting in the identification of 1797 peptides with high confidence by high resolution MS. These peptides covered 959 individual proteins constituting the largest collection of BALF proteins to date. High throughput monitoring profiles of this extensive collection of BALF proteins will facilitate the discovery and validation of biomarkers that would elucidate pathogenic or adaptive responses of the lungs upon toxic insults.

Residue formations of phosphorus hydride polymers and phosphorus oxyacids during phosphine gas fumigations of stored products.

With the extent of international usage and the critical role phosphine gas (PH3) plays in commercial pest control, identification of the residual components deposited during fumigation is mandatory. It has been postulated that these infrequent residues are primarily composed of phosphoric acid or reduced forms of phosphoric acid [hypophosphorous acid (H3PO2) and phosphorous acid (H3PO3)], due to the oxidative degradation of phosphine. Using environmental scanning electron microscopy, gas phase Fourier transform infrared spectroscopy, and X-ray fluorescence spectroscopy, the structural elucidation and formation mechanism of the yellow amorphous polyhydric phosphorus polymers (P(x)H(y)) that occur in addition to the lower oxyacids of phosphorus in residues deposited during PH3 fumigations of select tobacco commodities are explored. This research determined that nitric oxide gas (or nitrogen dioxide) initiates residue formation of phosphorus hydride polymers and phosphorus oxyacids during PH3 fumigations of stored products.

Determination of the relative energies of activation for the dissociation of aromatic versus aliphatic phosphopeptides by ESI-FTICR-MS and IRMPD.

Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (ESI-FTICR-MS) coupled with infrared multiphoton dissociation (IRMPD) is potentially a powerful method for rapid phosphopeptide mapping of complex proteolytic digests. The dissociation of deprotonated phosphopeptides by IRMPD is energetically favorable over unmodified deprotonated peptides because of a lower energy of activation and a higher internal energy under identical irradiation conditions. The energies of activation for dissociation are determined for model peptides phosphorylated on an aliphatic side chain (serine) and an aromatic side chain (tyrosine). The determination of phosphorylation location provides important biochemical information identifying the kinase involved in specific phosphorylation mechanisms. The data presented in this manuscript also support the theory that for phosphopeptides, the phosphate moiety's P-O stretch is in direct resonance with the infrared laser (10.6 microm), thus increasing the relative absorptivity of the modified species. A greater extinction coefficient affords more extensive photon absorption and subsequently a greater internal energy at the rapid exchange limit.

Dual-micro-ESI source for precise mass determination on a quadrupole time-of-flight mass spectrometer for genomic and proteomic applications.

A universal dual-electrospray (ESI) source is demonstrated on a quadrupole orthogonal-accelerated time-of-flight mass spectrometer (Q-ToF-MS) for both genomic and proteomic applications. This facile source modification enables internal calibration for consistent mass measurements by a mainstream MS platform and requires no mixing of analyte and calibrant prior to ion formation. In this report, the dual-sprayer is demonstrated in the negative-ion mode for internal calibration of polymerase chain reaction (PCR) amplicons generated from synthetic and genomic templates as well as a proteolytic digest of a naturally phosphorylated protein. For all PCR amplicons, experimentally determined average mass measurements are well within the instrument specifications of better than 0.01%. For the proteolytic fragments of the phosphoprotein, average mass errors of the isotopically resolved peptides are better than 10 ppm.

Gas-phase ion unimolecular dissociation for rapid phosphopeptide mapping by IRMPD in a Penning ion trap: an energetically favored process.

This communication discusses the efficient detection of the most important and common protein modification, phosphorylation, using ESI-FTICR-MS and IRMPD. Preliminary studies have demonstrated that within a complex protein digest, all phosphopeptides can be identified by a single IR laser irradiation event due to preferential dissociation of the modified peptides. This research demonstrates that the energy of activation for dissociation of the phosphopeptides is lower than that of the unmodified analogues providing the basis for the success of this technique. However, the P-O stretch (9.6-11 mum or 1042-909 cm-1) of this posttranslational modification is in direct resonance with the CO2 IR laser (10.6 mum or 943 cm-1) used for IRMPD. Therefore, the vibrational frequency of the phosphate moiety may be an additional factor in the rapid first-order decay of phosphopeptides. Based upon the energetics of dissociation discussed in this manuscript, IRMPD of ions in a Penning ion trap is an ideal platform for rapid phosphopeptide mapping.

Solution composition and thermal denaturation for the production of single-stranded PCR amplicons: piperidine-induced destabilization of the DNA duplex?

Strategies to produce single-stranded PCR amplicons for detection by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) were investigated using modified electrospray solutions and by thermally denaturing the duplex structures with a resistively heated electrospray ionization source. A synthetic 20-mer oligonucleotide annealed to its complementary strand was used as a model system for initial experiments. Electrospray solutions were altered by varying the relative proportion of aqueous phase in efforts to induce destabilization of the double helix. When the electrospray solution contains a 25% aqueous content, the 20-mer oligonucleotide is detected in its double-stranded form. Increasing the proportion of aqueous phase in the electrospray solution to 60% destabilized the double helix, resulting in the detection of only single-stranded species. This strategy was extended to an 82-bp polymerase chain reaction (PCR) product derived from the human tyrosine hydroxylase gene (HUMTH01). In efforts to destabilize the 82-bp PCR product, electrospray solutions reaching 70% aqueous content were necessary to promote the detection of only single-stranded amplicons. Implementation of the resistively heated transfer line and an electrospray solution in which the oligonucleotide is on the threshold of duplex stability allowed for double-stranded and single-stranded species to be generated from the same ESI solutions at both ambient and elevated transfer line temperatures, respectively, without disruption of the electrospray process. The volatile base piperidine, present at 20 mM concentrations in the electrospray solution, was found to play a critical role in the formation of single-stranded species at the higher aqueous percentages and a duplex destabilization mechanism has been proposed.