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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. [ABSTRACT FROM AUTHOR]

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 1 H 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.

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.

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 1 H 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.

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 (α fb ) e-liquid , with a possible range of 0 < (α fb ) e-liquid < 1. For e-liquids of an early design, (α fb ) 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": (α fb ) e-liquid values significantly less than 1 are now common. A framework is developed for predicting α fb values in a given medium based on the following: (1) acid/nicotine ratios and (2) overall acid + nicotine protonation constant ( K oa ) values. This framework is required for understanding (1) e-liquid design in regard to how acid additives affect (α fb ) e-liquid values, and (2) why (α fb ) e-liquid values cannot, in general, be measured by any method that involves significant dilution with water.

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 (α fb ) ranges from 0 to 1. Herein, we report α fb for IQOS aerosols by exchange-averaged 1 H NMR chemical shifts of the nicotine methyl protons in bulk aerosol and verified by headspace-solid phase microextraction-gas chromatography-mass spectrometry. The α fb ≈ 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 α fb for these products, however, their methods were subject to error due to solvent perturbation.

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 α fb , with 0 ≤ α fb ≤ 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 α fb is relevant regarding abuse liability. Previous attempts to determine α fb in electronic cigarette liquids and vapor have been flawed. We employed the exchange-averaged 1 H NMR chemical shifts of nicotine to determine α fb 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 ≤ α fb ≤ 0.84. The α fb 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 α fb  (for ease of inhalation) are likely to be particularly problematic for public health.