Effects of E-Cigarette Flavor Enhancing Capsules on Inhalable Aerosols.

The flavor of inhaled e-cigarette aerosols may be augmented using crushable flavor capsules added to e-cigarettes. For example, Puff Krush contains breakable flavor capsules in a sorbent material. The capsules are crushed, and then, aerosol passes through the saturated sorbent material before inhalation. Herein, we used NMR and GC-MS to identify the capsule medium chain triglyceride (MCT) solvent and flavorants in selected Puff Krush flavor capsules and then determined which molecules from the capsule transfer into aerosols. MCTs from the Puff Krush were not found in the aerosols, and ∼50% of Puff Krush flavorants transferred into the aerosol upon vaping.

Kinetics of Aldehyde Flavorant-Acetal Formation in E-Liquids with Different E-Cigarette Solvents and Common Additives Studied by 1H NMR Spectroscopy.

Flavorants, nicotine, and organic acids are common additives found in the e-liquid carrier solvent, propylene glycol (PG) and/or glycerol (GL), at various concentrations. Some of the most concentrated and prevalent flavorants in e-liquids include trans-cinnamaldehyde, vanillin, and benzaldehyde. Aldehyde flavorants have been shown to react with PG and GL to form flavorant-PG and -GL acetals that have unique toxicity properties in e-liquids before aerosolization. However, there is still much that remains unknown about the effects of different e-cigarette solvents, water, nicotine, and organic acids on the rate of acetalization in e-liquids. We used 1H NMR spectroscopy to determine the first-order initial rate constant, half-life, and % acetal formed at equilibrium for flavorant-acetal formation in simulated e-liquids. Herein, we report that acetalization generally occurs at a faster rate and produces greater yields in e-liquids with higher ratios of GL (relative to PG). trans-Cinnamaldehyde acetals formed the fastest in 100% PG-simulated e-liquids, followed by benzaldehyde and vanillin based on their half-lives and rate constants. The acetal yield was greatest for benzaldehyde in PG e-liquids, followed by trans-cinnamaldehyde and vanillin. Acetalization in PG e-liquids containing aldehyde flavorants was inhibited by water and nicotine but catalyzed by benzoic acid. Flavorant-PG acetal formation was generally delayed in the presence of nicotine, even if benzoic acid was present at 2-, 4-, or 10-fold the nicotine concentration, as compared to the PG e-liquids with 2.5 mg/mL flavorant. Thus, commercial e-liquids with aldehyde flavorants containing a higher GL ratio (relative to PG), little water, no nicotine, nicotine with excess organic acids, or organic acids without nicotine would undergo acetalization the fastest and with the highest yield. Many commercial e-liquids must therefore contain significant amounts of flavorant acetals.

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.

Fragment-based screening by protein-detected NMR spectroscopy.

Fragment-based drug discovery (FBDD) identifies low molecular weight compounds that can be developed into ligands with high affinity and selectivity for therapeutic targets. Screening fragment libraries (<10,000 molecules) with biophysical techniques against macromolecules provides information about novel chemical spaces that bind the macromolecule and scaffolds that can be modified to increase potency. A fragment-screening pipeline requires a standardized protocol for target selection, library assembly and maintenance, library screening, and hit validation to ensure hit integrity. Herein, the fundamental aspects of a fragment screening pipeline-focusing on protein-detected NMR data collection and analysis-are discussed in detail for researchers to use as a resource in their FBDD projects. Selected screening targets must undergo rigorous stability and buffer testing by NMR spectroscopy to ensure the protein structure is stable for the entire screen. Biophysical instrumentation that rapidly measures protein thermostability is helpful in buffer screening. Molecules in fragment libraries are analyzed computationally and physically, stored at appropriate temperatures, and multiplexed in well plates for library conservation. The screening protocol is streamlined using liquid handling robotics for sample preparation and customized Python scripts for protein-detected NMR data analysis. Molecules identified from the screen are titrated to determine their binding site(s) and Kd values and confirmed with an orthogonal biophysical assay. This detailed FBDD screening pipeline developed by the Program in Chemical Biology at the Medical College of Wisconsin has successfully screened many unrelated target proteins to identified novel molecules that selectively bind to these target proteins.