Determination of Nicotine Protonation State in E-Liquids by Low-Resolution Benchtop NMR Spectroscopy.

Over several years, e-liquids with "nicotine salts" have gained considerable popularity. These e-liquids have a low pH, at which nicotine occurs mostly in its monoprotonated form. Manufacturers usually accomplish this by the addition of an organic acid, such as levulinic acid, benzoic acid, or lactic acid. Nicotine in its protonated form can be more easily inhaled, enhancing the addictiveness and attractiveness of products. Several techniques have been described for measuring the protonation state of nicotine in e-liquids. However, nuclear magnetic resonance (NMR) spectroscopy is particularly suited for this purpose because it can be performed on unaltered e-liquids. In this article, we demonstrate the suitability of a benchtop NMR (60 MHz) instrument for determining the protonation state of nicotine in e-liquids. The method is subsequently applied to measure the protonation state of 33 commercially available e-liquids and to investigate whether the vaping process alters the protonation state of nicotine. For this purpose, the protonation state in the condensed aerosol obtained by automated vaping of different e-liquids was compared with that of the original e-liquids. Two distinct populations were observed in the protonation state of nicotine in commercial e-liquids: free-base (fraction of free-base nicotine αfb > 0.80) and protonated (αfb < 0.40). For 30 e-liquids out of 33, the information on the packaging regarding the presence of nicotine salt was in agreement with the observed protonation state. Three e-liquids contained nicotine salt, even though this was not stated on the packaging. Measuring the protonation state of nicotine before and after (machine) vaping revealed that the protonation state of e-liquids is not affected by vaping. In conclusion, it is possible to determine the nicotine protonation state with the described method. Two clusters can be distinguished in the protonation state of commercial e-liquids, and the protonation state of nicotine remains unchanged after vaping.

Variations in cigarette brand characteristics: can consumers tell the difference?

OBJECTIVES: Sensory experience is an important determinant of smoking initiation, brand choice and harm perception, but little is known about how cigarette design shapes sensory experience. This study reports which variations in tobacco blend and design characteristics available on the market are likely to be perceived as different by consumers. METHODS: Truth Tobacco Industry Documents was reviewed for studies showing noticeable sensory differences resulting from variations in tobacco blend and design characteristics. These differences were compared with tobacco product data as available in the Dutch section of the European Common Entry Gate (EU-CEG) system on 30 April 2020. RESULTS: Industry documents identified discrimination thresholds for ventilation, pressure drop, tobacco weight, filter length, and tar and nicotine levels in smoke while evidence for other design characteristics was less conclusive. In the 103 different cigarette varieties in the EU-CEG database, five main types of cigarettes could be identified by principal component analysis, differing in (combinations of) design characteristics. The most significant differences between brand varieties were tar, nicotine and carbon monoxide emissions and associated parameters filter ventilation, filter length, cigarette length and tobacco weight. CONCLUSIONS: While some clusters of brand varieties provided a noticeably different product for consumers, in many cases design differences within these clusters did not exceed the expected discrimination threshold. This indicates that many products on the market are not discernibly different for consumers, and that proliferation of brand varieties has a non-sensory purpose, such as marketing. Policy makers should consider limiting available brand varieties and regulating design characteristics to reduce product appeal.

Aldehyde and Volatile Organic Compound Yields in Commercial Cigarette Mainstream Smoke Are Mutually Related and Depend on the Sugar and Humectant Content in Tobacco.

INTRODUCTION: The World Health Organization (WHO) Framework Convention on Tobacco control recognizes the need for tobacco product regulation. In line with that, the WHO Study Group on Tobacco Product Regulation (TobReg) proposed to regulate nine toxicants in mainstream cigarette smoke, including aldehydes, volatile organic compounds (VOCs), and carbon monoxide (CO). We analyzed their relations in 50 commercially available cigarette brands, using two different smoking regimes, and their dependence on sugar and humectant concentrations in tobacco filler. METHODS: We measured sugar and humectant in tobacco filler and aldehydes, VOCs, and tar, nicotine, and CO (TNCO) in mainstream smoke. The general statistics, correlations between emission yields, and correlations between contents and emissions yields were determined for these data. RESULTS: For aldehydes, several significant correlations were found with precursor ingredients in unburnt tobacco when smoked with the Intense regime, most prominently for formaldehyde with sucrose, glucose, total sugars, and glycerol. For VOCs, 2,5-dimethylfuran significantly correlates with several sugars under both International Standards Organization (ISO) and Intense smoking conditions. A correlation network visualization shows connectivity between a sugar cluster, an ISO cluster, and an Intense cluster, with Intense formaldehyde as a central highest connected hub. CONCLUSIONS: Our multivariate analysis showed several strong connections between the compounds determined. The toxicants proposed by WHO, in particular, formaldehyde, can be used to monitor yields of other toxicants under Intense conditions. Emissions of formaldehyde, acetaldehyde, acrolein, and 2,5-dimethylfuran may decrease when sugar and humectants contents are lowered in tobacco filler. IMPLICATIONS: Our findings suggest that the aldehydes and VOCs proposed by TobReg are a representative selection for smoke component market monitoring purposes. In particular, formaldehyde yields may be useful to monitor emissions of other toxicants under Intense conditions. Since the most and strongest correlations were observed with the Intense regime, policymakers are advised to prescribe this regime for regulatory purposes. Policymakers should also consider sugars and humectants contents as targets for future tobacco product regulations, with the additional advantage that consumer acceptance of cigarette smoke is proportional to their concentrations in the tobacco blend.

Cigarette Filter Ventilation and Smoking Protocol Influence Aldehyde Smoke Yields.

The WHO study group on tobacco product regulation (TobReg) advised regulating and lowering toxicant levels in cigarette smoke. Aldehydes are one of the chemical classes on the TobReg smoke toxicants priority list. To provide insight in factors determining aldehyde yields, the levels of 12 aldehydes in mainstream cigarette smoke of 11 Dutch brands were quantified. Variations in smoking behavior and cigarette design affecting human exposure to aldehydes were studied by using four different machine testing protocols. Machine smoking was based on the International Standardization Organization (ISO) and Health Canada Intense (HCI) regime, both with and without taping the filter vents. The 11 cigarette brands differed in (i) design and blend characteristics; (ii) tar, nicotine, and carbon monoxide (TNCO) levels; (iii) popularity; and (iv) manufacturer. Cigarette smoke was trapped on a Cambridge filter pad and carboxen cartridge. After being dissolved in methanol/CS2 and derivatization with DNPH, the aldehyde yields were determined using HPLC-DAD. Using an intense smoking regime (increased puff volume, shorter puff interval) significantly increased aldehyde yields, following the pattern: ISO < ISO-taped < HCI-untaped < HCI. For all of the regimes, acetaldehyde and acrolein yields were strongly correlated ( r = 0.804). The difference in TNCO and aldehyde levels between regular and highly ventilated low-TNCO cigarettes (as measured using ISO) diminished when smoking intensely; this effect is stronger when combined with taping filter vents. The highly ventilated low-TNCO brands showed six times more aldehyde production per mg nicotine for the intense smoking regimes. In conclusion, acetaldehyde and acrolein can be used as representatives for the class of volatile aldehydes for the different brands and smoking regimes. The aldehyde-to-nicotine ratio increased when highly ventilated cigarettes were smoked intensely, similar to real smokers. Thus, a smoker of highly ventilated low-TNCO cigarettes has an increased potential for higher aldehyde exposures compared to a smoker of regular cigarettes.

Smoking-Associated Exposure of Human Primary Bronchial Epithelial Cells to Aldehydes: Impact on Molecular Mechanisms Controlling Mitochondrial Content and Function.

Chronic obstructive pulmonary disease (COPD) is a devastating lung disease primarily caused by exposure to cigarette smoke (CS). During the pyrolysis and combustion of tobacco, reactive aldehydes such as acetaldehyde, acrolein, and formaldehyde are formed, which are known to be involved in respiratory toxicity. Although CS-induced mitochondrial dysfunction has been implicated in the pathophysiology of COPD, the role of aldehydes therein is incompletely understood. To investigate this, we used a physiologically relevant in vitro exposure model of differentiated human primary bronchial epithelial cells (PBEC) exposed to CS (one cigarette) or a mixture of acetaldehyde, acrolein, and formaldehyde (at relevant concentrations of one cigarette) or air, in a continuous flow system using a puff-like exposure protocol. Exposure of PBEC to CS resulted in elevated IL-8 cytokine and mRNA levels, increased abundance of constituents associated with autophagy, decreased protein levels of molecules associated with the mitophagy machinery, and alterations in the abundance of regulators of mitochondrial biogenesis. Furthermore, decreased transcript levels of basal epithelial cell marker KRT5 were reported after CS exposure. Only parts of these changes were replicated in PBEC upon exposure to a combination of acetaldehyde, acrolein, and formaldehyde. More specifically, aldehydes decreased MAP1LC3A mRNA (autophagy) and BNIP3 protein (mitophagy) and increased ESRRA protein (mitochondrial biogenesis). These data suggest that other compounds in addition to aldehydes in CS contribute to CS-induced dysregulation of constituents controlling mitochondrial content and function in airway epithelial cells.

Improving the Analysis of E-Cigarette Emissions: Detecting Human "Dry Puff" Conditions in a Laboratory as Validated by a Panel of Experienced Vapers.

INTRODUCTION: E-cigarette product regulation requires accurate analyses of emissions. User behavior, including device power setting selection, should be mimicked closely when generating e-cigarette emissions in a laboratory. Excessively high power settings result in an adverse burnt off-taste, called "dry puff flavor". This should be avoided because it results in an overestimation of toxicant levels (especially certain carbonyls). This study presents a human volunteer-validated approach to detect excessively high e-cigarette power settings by HPLC-DAD (high-performance liquid chromatography-diode array detection) carbonyl analysis. METHODS: Thirteen experienced e-cigarette users evaluated whether the "dry puff flavor" was present at different power settings (10 W-25 W), recording their assessment on a 100-unit visual analog scale (VAS). They assessed e-cigarettes equipped with 1.2 Ω or 1.6 Ω coils containing menthol, vanilla or fruit-flavored e-liquids. In a machine-vaping experiment, emissions from the same liquid/coil/power setting combinations were subjected to HPLC-DAD analysis of dinitrophenol hydrazine (DNPH)-derivatized carbonyls, such as lactaldehyde and formaldehyde. A simple algorithm, based on the cutoff values for each marker, was applied to relate the dry puff flavor (as assessed by the human volunteers) to the laboratory measurements. RESULTS: Eleven carbonyl compounds were found to agree with the human assessments. Based on the amounts of these compounds in the emissions, the dry-puff flavor did match at all combinations of e-liquids and coils examined. Dry-puff flavor was observed at different power levels with the different liquids tested. CONCLUSIONS: The described method can detect dry puff conditions and is therefore a useful tool to ensure user-relevant conditions in laboratory analyses of e-cigarette emissions. IMPLICATIONS: This study improves the chemical analysis of e-cigarette emissions. It offers a method to select an appropriate (i.e., user-relevant) power setting for e-cigarettes, which is a critical parameter for emission analysis and therefore important for regulatory purposes and risk assessments. Compared to the approach of using human volunteers to select appropriate power settings for different products by taste, the described method is cheaper, faster, more practical and more ethical.