Effects of Common e-Liquid Flavorants and Added Nicotine on Toxicant Formation during Vaping Analyzed by 1H NMR Spectroscopy.

A broad variety of e-liquids are used by e-cigarette consumers. Additives to the e-liquid carrier solvents, propylene glycol and glycerol, often include flavorants and nicotine at various concentrations. Flavorants in general have been reported to increase toxicant formation in e-cigarette aerosols, yet there is still much that remains unknown about the effects of flavorants, nicotine, and flavorants + nicotine on harmful and potentially harmful constituents (HPHCs) when aerosolizing e-liquids. Common flavorants benzaldehyde, vanillin, benzyl alcohol, and trans-cinnamaldehyde have been identified as some of the most concentrated flavorants in some commercial e-liquids, yet there is limited information on their effects on HPHC formation. E-liquids containing flavorants + nicotine are also common, but the specific effects of flavorants + nicotine on toxicant formation remain understudied. We used 1H NMR spectroscopy to evaluate HPHCs and herein report that benzaldehyde, vanillin, benzyl alcohol, trans-cinnamaldehyde, and mixtures of these flavorants significantly increased toxicant formation produced during e-liquid aerosolization compared to unflavored e-liquids. However, e-liquids aerosolized with flavorants + nicotine decreased the HPHCs for benzaldehyde, vanillin, benzyl alcohol, and a "flavorant mixture" but increased the HPHCs for e-liquids containing trans-cinnamaldehyde compared to e-liquids with flavorants and no nicotine. We determined how nicotine affects the production of HPHCs from e-liquids with flavorant + nicotine versus flavorant, herein referred to as the "nicotine degradation factor". Benzaldehyde, vanillin, benzyl alcohol, and a "flavorant mixture" with nicotine showed lower HPHC levels, having nicotine degradation factors <1 for acetaldehyde, acrolein, and total formaldehyde. HPHC formation was most inhibited in e-liquids containing vanillin + nicotine, with a degradation factor of ∼0.5, while trans-cinnamaldehyde gave more HPHC formation when nicotine was present, with a degradation factor of ∼2.5 under the conditions studied. Thus, the effects of flavorant molecules and nicotine are complex and warrant further studies on their impacts in other e-liquid formulations as well as with more devices and heating element types.

Ratio of Propylene Glycol to Glycerol in E-Cigarette Reservoirs Is Unchanged by Vaping As Determined by 1H NMR Spectroscopy.

E-cigarette liquids (e-liquids) contain propylene glycol (PG) and/or glycerol (GL) to deliver flavorants/nicotine. It has recently been suggested that the PG:GL ratio in e-cigarette reservoirs changes during vaping, leaving almost entirely GL after aerosolizing much of a 30:70 PG:GL mixture. To evaluate this directly, we analyzed e-liquids from e-cigarettes before and after aerosolization using 4 different coils, and aerosol samples generated using high and low e-liquid levels. The PG:GL ratios of initial and final e-liquids and aerosol samples were comparable. This is important because a large change in e-liquid composition could substantially alter the aerosol profile during a vaping session.

Determination of (R)-(+)- and (S)-(-)-Nicotine Chirality in Puff Bar E-Liquids by 1H NMR Spectroscopy, Polarimetry, and Gas Chromatography-Mass Spectrometry.

Tobacco products generally contain tobacco-derived nicotine (TDN; having ∼99+% (S)-(-)-nicotine). Recent United States regulation has led some producers to transition to synthetic ("tobacco-free") nicotine. For example, Puff Bar is now marketed with tobacco-free nicotine (TFN; presumed to be racemic). To evaluate the claim that these new products contain TFN, we evaluated the presence of the two nicotine optical isomers by 1H NMR spectroscopy, polarimetry, and gas chromatography-mass spectrometry. Older Puff Bars were found to contain (S)-(-)-nicotine, and newer "TFN" Puff Bars were found to contain both (R)-(+) and (S)-(-) isomers-indicating TFN, albeit with slightly more of the (S)-(-)-nicotine form.

Free-Base Nicotine Fraction αfb in Non-Aqueous versus Aqueous Solutions: Electronic Cigarette Fluids Without versus With Dilution with Water.

An important design aspect of electronic cigarettes ("e-cigarettes") is the nature of the acid/base chemistry in the e-liquid phase. E-liquids having formulations similar to those of early products are mixes of propylene glycol/glycerol (PG/GL) plus free-base (fb) nicotine and (usually) flavor chemicals that are either rather weak or non-acid/base actors in PG/GL. The fraction of nicotine in the fb form is denoted (αfb)e-liquid, with a possible range of 0 < (αfb)e-liquid < 1. For e-liquids of an early design, (αfb)e-liquid ≈ 1. Because e-cigarette aerosols high in fb nicotine are harsh upon inhalation, many commercial e-liquids now also contain variable levels of an acid additive (e.g., benzoic acid, levulinic acid, etc.) to protonate the nicotine and form dissolved "nicotine salts": (αfb)e-liquid values significantly less than 1 are now common. A framework is developed for predicting αfb values in a given medium based on the following: (1) acid/nicotine ratios and (2) overall acid + nicotine protonation constant (Koa) values. This framework is required for understanding (1) e-liquid design in regard to how acid additives affect (αfb)e-liquid values, and (2) why (αfb)e-liquid values cannot, in general, be measured by any method that involves significant dilution with water.

Nicotine in tobacco product aerosols: 'It's déjà vu all over again'.

INTRODUCTION: The distribution of nicotine among its free-base (fb) and protonated forms in aerosolised nicotine affects inhalability. It has been manipulated in tobacco smoke and now in electronic cigarettes by the use of acids to de-freebase nicotine and form 'nicotine salts'. METHODS: Measurements on electronic cigarette fluids (e-liquids) were carried out to determine (1) the fraction of nicotine in the free-base form (αfb) and (2) the levels of organic acid(s) and nicotine. Samples included JUUL 'pods', 'look-a-like/knock-off' pods and some bottled 'nicotine salt' and 'non-salt' e-liquids. RESULTS: αfb= 0.12 ±0.01 at 40°C (≈ 37°C) for 10 JUUL products, which contain benzoic acid; nicotine protonation is extensive but incomplete. DISCUSSION: First-generation e-liquids have αfb ≈ 1. At cigarette-like total nicotine concentration (Nictot) values of ~60 mg/mL, e-liquid aerosol droplets with αfb≈ 1 are harsh upon inhalation. The design evolution for e-liquids has paralleled that for smoked tobacco, giving a 'déjà vu' trajectory for αfb. For 17th-century 'air-cured' tobacco, αfb in the smoke particles was likely ≥ 0.5. The product αfbNictot in the smoke particles was high. 'Flue-curing' retains higher levels of leaf sugars, which are precursors for organic acids in tobacco smoke, resulting in αfb ≈ 0.02 and lowered harshness. Some tobacco cigarette formulations/designs have been adjusted to restore some nicotine sensory 'kick/impact' with αfb≈ 0.1, as for Marlboro. Overall, for tobacco smoke, the de-freebasing trajectory was αfb ≥ 0.5 → ~0 →~0.1, as compared with αfb= ~1 →~0.1 for e-cigarettes. For JUUL, the result has been, perhaps, an optimised, flavoured nicotine delivery system. The design evolution for e-cigarettes has made them more effective as substitutes to get smokers off combustibles. However, this evolution has likely made e-cigarette products vastly more addictive for never-smokers.

Sucralose-Enhanced Degradation of Electronic Cigarette Liquids during Vaping.

Electronic cigarette liquids (e-liquids) with sweetener additives such as sucralose, a synthetic chlorinated disaccharide, are popular among some e-cigarette consumers; sucralose can be added either by the manufacturer or by the consumer. The prevalence of sucralose in commercial e-liquids is not known, nor is the typical concentration of sucralose when present; labels are not required to disclose ingredient information. Here, we report the effects of sucralose on e-liquid degradation upon e-cigarette vaping as studied using 1H NMR spectroscopy, ion chromatography, and gas chromatography coupled with detection by mass spectrometry or flame ionization detector. Sucralose was found to be subject to degradation when included in propylene glycol + glycerol based e-liquids and vaped; the presence of sucralose in the e-liquids also resulted in altered and enhanced solvent degradation. In particular, production of aldehydes (carbonyls) and hemiacetals (which have implications for health) was enhanced, as demonstrated by 1H NMR. The presence of sucralose at 0.03 mol % (0.14 wt %) in an e-liquid also resulted in production of potentially harmful organochlorine compounds and catalyzed the cyclization of aldehydes with solvents to acetals upon vaping; the presence of chloride in e-liquid aerosols was confirmed by ion chromatography. Quantities of sucralose as low as 0.05 mol % (0.24 wt %) in e-liquids lead to significant production of solvent degradation products.

Free-Base Nicotine Is Nearly Absent in Aerosol from IQOS Heat-Not-Burn Devices, As Determined by 1H NMR Spectroscopy.

Heat-not-burn products, eg, I quit ordinary smoking (IQOS), are becoming popular alternative tobacco products. The nicotine aerosol protonation state has addiction implications due to differences in absorption kinetics and harshness. Nicotine free-base fraction (αfb) ranges from 0 to 1. Herein, we report αfb for IQOS aerosols by exchange-averaged 1H NMR chemical shifts of the nicotine methyl protons in bulk aerosol and verified by headspace-solid phase microextraction-gas chromatography-mass spectrometry. The αfb ≈ 0 for products tested; likely a result of proton transfer from acetic acid and/or other additives in the largely aqueous aerosol. Others reported higher αfb for these products, however, their methods were subject to error due to solvent perturbation.

Free-Base Nicotine Determination in Electronic Cigarette Liquids by 1H NMR Spectroscopy.

E-liquids usually contain significant nicotine, which will exist primarily in two forms, monoprotonated and free-base, the proportions of which are alterable through the effective pH of the medium. The fraction of nicotine in the free-base form is αfb, with 0 ≤ αfb ≤ 1. When dosed via aerosol, the two nicotine forms have different mechanisms and kinetics of delivery, as well as differing implications for harshness of the inhaled aerosol, so αfb is relevant regarding abuse liability. Previous attempts to determine αfb in electronic cigarette liquids and vapor have been flawed. We employed the exchange-averaged 1H NMR chemical shifts of nicotine to determine αfb in samples of e-liquids. This method is rapid and direct and can also be used with collected aerosol material. The e-liquids tested were found to have 0.03 ≤ αfb ≤ 0.84. The αfb values in collected aerosol liquid samples were highly correlated with those for the parent e-liquids. E-liquids designed to combine high total nicotine level (addictive delivery) with low αfb (for ease of inhalation) are likely to be particularly problematic for public health.

Quantification of PARP7 Protein Levels and PARP7 Inhibitor Target Engagement in Cells Using a Split Nanoluciferase System.

PARP7 is an enzyme that catalyzes mono-ADP-ribosylation (MARylation), is a critical regulator of type I interferon signaling, and has emerged as an immune-oncology drug candidate. PARP7 is a labile protein that is regulated in a proteasome-dependent manner. Indeed, endogenous PARP7 levels are undetectable by western blot in most cells. Intriguingly, treatment of cells with orthosteric small molecule inhibitors of PARP7 can increase endogenous PARP7 protein to detectable levels. This characteristic of PARP7 inhibitors could potentially be exploited to assess target engagement-and thus cellular efficacy-of PARP7 inhibitors; however, no method exists to quantitatively monitor endogenous PARP7 levels in a high-throughput manner. In this protocol, we describe an assay using a split Nanoluciferase (NanoLuc) system for quantifying endogenous PARP7 protein levels and PARP7 inhibitor target engagement in cells in a 96-well plate format. We show that this assay can be used to quantify PARP7 protein levels under various cellular treatments and can assess cellular PARP7 inhibitor target engagement. We envision this split NanoLuc PARP7 assay can be used not only for evaluating the cellular efficacy of PARP7 inhibitors in a high-throughput manner but also for uncovering the mechanisms regulating PARP7 protein levels in cells.

Structurally distinct PARP7 inhibitors provide new insights into the function of PARP7 in regulating nucleic acid-sensing and IFN-ß signaling.

The mono-ADP-ribosyltransferase PARP7 has emerged as a key negative regulator of cytosolic NA-sensors of the innate immune system. We apply a rational design strategy for converting a pan-PARP inhibitor into a potent selective PARP7 inhibitor (KMR-206). Consistent with studies using the structurally distinct PARP7 inhibitor RBN-2397, co-treatment of mouse embryonic fibroblasts with KMR-206 and NA-sensor ligands synergistically induced the expression of the type I interferon, IFN-ß. In mouse colon carcinoma (CT-26) cells, KMR-206 alone induced IFN-ß. Both KMR-206 and RBN-2397 increased PARP7 protein levels in CT-26 cells, demonstrating that PARP7's catalytic activity regulates its own protein levels. Curiously, treatment with saturating doses of KMR-206 and RBN-2397 achieved different levels of PARP7 protein, which correlated with the magnitude of type I interferon gene expression. These latter results have important implications for the mechanism of action of PARP7 inhibitors and highlights the usefulness of having structurally distinct chemical probes for the same target.

Allosteric regulation of DNA binding and target residence time drive the cytotoxicity of phthalazinone-based PARP-1 inhibitors.

Allosteric coupling between the DNA binding site to the NAD+-binding pocket drives PARP-1 activation. This allosteric communication occurs in the reverse direction such that NAD+ mimetics can enhance PARP-1's affinity for DNA, referred to as type I inhibition. The cellular effects of type I inhibition are unknown, largely because of the lack of potent, membrane-permeable type I inhibitors. Here we identify the phthalazinone inhibitor AZ0108 as a type I inhibitor. Unlike the structurally related inhibitor olaparib, AZ0108 induces replication stress in tumorigenic cells. Synthesis of analogs of AZ0108 revealed features of AZ0108 that are required for type I inhibition. One analog, Pip6, showed similar type I inhibition of PARP-1 but was ∼90-fold more cytotoxic than AZ0108. Washout experiments suggest that the enhanced cytotoxicity of Pip6 compared with AZ0108 is due to prolonged target residence time on PARP-1. Pip6 represents a new class of PARP-1 inhibitors that may have unique anticancer properties.

Boiling points of the propylene glycol + glycerol system at 1 atmosphere pressure: 188.6-292 °C without and with added water or nicotine.

In electronic cigarettes ("electronic nicotine delivery systems", ENDS), mixtures of propylene glycol (PG) and/or glycerol (GL; aka "vegetable glycerin", VG) with nicotine are vaporized to create a nicotine-containing aerosol. For a given composition, the temperature required to boil the liquid at 1 atmosphere must be at least somewhat greater than the boiling point (BP). The use of ENDS is increasing rapidly worldwide, yet the BP characteristics of the PG + GL system have been characterized as the mixtures; here we re-do this, but significantly, also study the effects of added water and nicotine. BP values at 1 atmosphere pressure were measured over the full binary composition range. Fits based on the Gibbs-Konovalov theorem provide BP as a function of composition (by mole-percent, by weight-percent, and by volume-percent). BPs of PG + GL mixtures were then tested in the presence of additives such as water (2.5 and 5 mol% added) and nicotine (3 mol%). Water was found to decrease the BP of PG + GL mixtures significantly at all compositions tested, and nicotine was found to decrease the BP of PG + GL mixtures containing ~75 GL: 25 PG (by moles) or more. The effect of added water (5, 10, and 15 mol% added) on electronic cigarette degradation production (some aldehydes and formaldehyde hemiacetals) was examined and found to have no significant impact on solvent (PG or GL) degradation for the particular device used.

Benzene formation in electronic cigarettes.

BACKGROUND/OBJECTIVE: The heating of the fluids used in electronic cigarettes ("e-cigarettes") used to create "vaping" aerosols is capable of causing a wide range of degradation reaction products. We investigated formation of benzene (an important human carcinogen) from e-cigarette fluids containing propylene glycol (PG), glycerol (GL), benzoic acid, the flavor chemical benzaldehyde, and nicotine. METHODS/MAIN RESULTS: Three e-cigarette devices were used: the JUULTM "pod" system (provides no user accessible settings other than flavor cartridge choice), and two refill tank systems that allowed a range of user accessible power settings. Benzene in the e-cigarette aerosols was determined by gas chromatography/mass spectrometry. Benzene formation was ND (not detected) in the JUUL system. In the two tank systems benzene was found to form from propylene glycol (PG) and glycerol (GL), and from the additives benzoic acid and benzaldehyde, especially at high power settings. With 50:50 PG+GL, for tank device 1 at 6W and 13W, the formed benzene concentrations were 1.9 and 750 µg/m3. For tank device 2, at 6W and 25W, the formed concentrations were ND and 1.8 µg/m3. With benzoic acid and benzaldehyde at ~10 mg/mL, for tank device 1, values at 13W were as high as 5000 µg/m3. For tank device 2 at 25W, all values were ≤~100 µg/m3. These values may be compared with what can be expected in a conventional (tobacco) cigarette, namely 200,000 µg/m3. Thus, the risks from benzene will be lower from e-cigarettes than from conventional cigarettes. However, ambient benzene air concentrations in the U.S. have typically been 1 µg/m3, so that benzene has been named the largest single known cancer-risk air toxic in the U.S. For non-smokers, chronically repeated exposure to benzene from e-cigarettes at levels such as 100 or higher µg/m3 will not be of negligible risk.