Limonene Enantiomeric Ratios from Anthropogenic and Biogenic Emission Sources.

Emissions from volatile chemical products (VCPs) have been identified as contributors to air quality degradation in urban areas. Limonene can be a tracer compound for VCPs containing fragrances in densely populated regions, but limonene is also emitted from conifers that are planted in urban areas. This creates challenges for using limonene to estimate VCP emissions. In this study, the -/+ enantiomeric ratios of limonene from VCP and conifer emission sources were quantified to evaluate if this measurement could be used to aid in source apportionment and emission inventory development. Samples were analyzed using a gas chromatograph equipped with a chiral column and mass spectrometry. The results demonstrate that limonene exhibits distinct enantiomeric ratios when sourced from VCPs versus conifers. (+)-Limonene was dominant in VCP sources (>97%), which was not universally true for conifer sources. The results were compared to those of air samples collected outside at two locations and indoors. The levels of (-)-limonene in outdoor air in Irvine and Portland and in indoor air were 50%, 22%, and 4%, respectively. This suggests outdoor limonene had both VCP and plant emission sources while indoor air was dominated by VCP sources. This study demonstrates the potential utility of enantiomeric analysis for improving VCP emission estimates in urban areas.

Menthol and Other Flavor Chemicals in Cigarettes from Vietnam and the Philippines.

INTRODUCTION: Tobacco product flavors can increase product appeal, adolescent initiation and experimentation, and difficulty quitting. Flavored tobacco products are not restricted in Vietnam or the Philippines despite the high smoking prevalence among those 15 years of age and older (24% and 23%, respectively). There are no published reports to our knowledge on the levels of flavor chemicals in the cigarettes sold in these two countries. METHODS: Cigarettes were purchased in Vietnam (32 brand variants) and the Philippines (19 brand variants) during 2020. Chemical analyses gave the mg/filter, mg/rod, and mg/stick (= mg/(filter + rod)) values for 180 individual flavor chemicals. Values were calculated for menthol, clove-related compounds, and "other flavor chemicals" (OFCs). RESULTS: Five flavor groupings were found among the brand variants purchased in Vietnam: menthol + OFCs (n = 15), OFCs only (n = 8), nonflavored (n = 7), menthol + OFCs with a clove flavorant (n = 1) and menthol only (n = 1). Three flavor groupings were found among the brand variants purchased in the Philippines: menthol + OFCs (n = 10), nonflavored (n = 5), and menthol only (n = 4). CONCLUSIONS: A range of flavored cigarette products are being offered by tobacco companies in Vietnam and the Philippines, presumably to maximize cigarette sales. Regulation of flavor chemicals should be considered in these two countries. IMPLICATIONS: Article 9 of the WHO Framework Convention on Tobacco Control (FCTC), ratified by both Vietnam and the Philippines, states that "there is no justification for permitting the use of ingredients, such as flavoring agents, which help make tobacco products attractive." Flavors increase product appeal, adolescent initiation and experimentation, and difficulty quitting. These analyses found that cigarettes purchased in Vietnam and the Philippines contained menthol and other flavor chemicals. Tobacco companies are offering multiple flavor chemical profiles and nominally nonflavored versions in these countries; regulation of flavor chemicals should be considered in these two countries.

CFA-treated mice induce hyperalgesia in healthy mice via an olfactory mechanism.

BACKGROUND: Social interactions with subjects experiencing pain can increase nociceptive sensitivity in observers, even without direct physical contact. In previous experiments, extended indirect exposure to soiled bedding from mice with alcohol withdrawal-related hyperalgesia enhanced nociception in their conspecifics. This finding suggested that olfactory cues could be sufficient for nociceptive hypersensitivity in otherwise untreated animals (also known as "bystanders"). AIM: The current study addressed this possibility using an inflammation-based hyperalgesia model and long- and short-term exposure paradigms in C57BL/6J mice. MATERIALS & METHOD: Adult male and female mice received intraplantar injection of complete Freund's adjuvant (CFA) and were used as stimulus animals to otherwise naïve same-sex bystander mice (BS). Another group of untreated mice (OLF) was simultaneously exposed to the bedding of the stimulus mice. RESULTS: In the long-term, 15-day exposure paradigm, the presence of CFA mice or their bedding resulted in reduced von Frey threshold but not Hargreaves paw withdrawal latency in BS or OLF mice. In the short-term paradigm, 1-hr interaction with CFA conspecifics or 1-hr exposure to their bedding induced mechanical hypersensitivity in BS and OLF mice lasting for 3 hrs. Chemical ablation of the main olfactory epithelium prevented bedding-induced and stimulus mice-induced mechanical hypersensitivity. Gas chromatography-mass spectrometry (GC-MS) analysis of the volatile compounds in the bedding of experimental mice revealed that CFA-treated mice released an increased number of compounds indicative of disease states. DISCUSSION AND CONCLUSION: These results demonstrate that CFA-induced inflammatory pain can modulate nociception in bystander mice via an olfactory mechanism involving dynamic changes in volatile compounds detectable in the rodent bedding. SIGNIFICANCE: Social context can influence nociceptive sensitivity. Recent studies suggested involvement of olfaction in this influence. In agreement with this idea, the present study shows that the presence of mice with inflammatory pain produces nociceptive hypersensitivity in nearby conspecifics. This enhanced nociception occurs via olfactory cues present in the mouse bedding. Analysis of the bedding from mice with inflammatory pain identifies a number of compounds indicative of disease states. These findings demonstrate the importance of olfactory system in influencing pain states.

A synthetic coolant (WS-23) in disposable electronic cigarettes impairs cytoskeletal function in EpiAirway microtissues exposed at the air liquid interface.

The design of popular disposable electronic cigarettes (ECs) was analyzed, and the concentrations of WS-23, a synthetic coolant, in EC fluids were determined for 22 devices from 4 different brands. All products contained WS-23 in concentrations that ranged from 1.0 to 40.1 mg/mL (mean = 21.4 ± 9.2 mg/mL). To determine the effects of WS-23 on human bronchial epithelium in isolation of other chemicals, we exposed EpiAirway 3-D microtissues to WS-23 at the air liquid interface (ALI) using a cloud chamber that generated aerosols without heating. Proteomics analysis of exposed tissues revealed that the cytoskeleton was a major target of WS-23. BEAS-2B cells were exposed to WS-23 in submerged culture to validate the main results from proteomics. F-actin, which was visualized with phalloidin, decreased concentration dependently in WS-23 treated BEAS-2B cells, and cells became immotile in concentrations above 1.5 mg/mL. Gap closure, which depends on both cell proliferation and migration, was inhibited by 0.45 mg/mL of WS-23. These data show that WS-23 is being added to popular EC fluids at concentrations that can impair processes dependent on the actin cytoskeleton and disturb homeostasis of the bronchial epithelium. The unregulated use of WS-23 in EC products may harm human health.

Eugenol, menthol and other flavour chemicals in kreteks and 'white' cigarettes purchased in Indonesia.

BACKGROUND: Flavoured tobacco products are not restricted in Indonesia, a country with about 68 million adults who smoke. Most use clove-mixed tobacco cigarettes ('kreteks'); non-clove ('white') cigarettes are also available. Although the use of flavour chemicals has been identified by WHO as promoting tobacco use, little has been reported for Indonesia about the levels of flavourants in either kreteks or 'white cigarettes'. METHODS: 22 kretek brand variants and nine 'white' cigarette brand variants were purchased in Indonesia during 2021/2022; one of the kretek packs contained three colour-coded variants, giving a total sample number of 24 for the kreteks. Chemical analyses gave the mg/stick (=mg/(filter+rod)) values for 180 individual flavour chemicals that included eugenol (a clove-flavoured compound), four other clove-related compounds and menthol. RESULTS: Eugenol was present at significant levels in all 24 kreteks (2.8-33.8 mg/stick), but was essentially absent in all of the cigarettes. Menthol was present in 14 of 24 kreteks, with levels ranging from 2.8 to 12.9 mg/stick, and in five of the nine cigarettes, with levels ranging from 3.6 to 10.8 mg/stick. Other flavour chemicals were also found in many of the kretek and cigarette samples. CONCLUSIONS: In this small sample, we found numerous variations of flavoured tobacco products offered by multinational and national companies in Indonesia. Given the body of evidence that flavours make tobacco products more appealing, regulation of clove-related compounds, menthol and other flavour chemicals should be considered in Indonesia.

Exposure, Retention, Exhalation, Symptoms, and Environmental Accumulation of Chemicals During JUUL Vaping.

Little is known about the chemical exposures that electronic cigarette (EC) users receive and emit during JUUL vaping and if exposures produce symptoms dose dependently. This study examined chemical exposure (dose), retention, symptoms during vaping, and the environmental accumulation of exhaled propylene glycol (PG), glycerol (G), nicotine, and menthol in a cohort of human participants who vaped JUUL "Menthol" ECs. We refer to this environmental accumulation as "EC exhaled aerosol residue" (ECEAR). Chemicals were quantified using gas chromatography/mass spectrometry in JUUL pods before and after use, lab-generated aerosols, human exhaled aerosols, and in ECEAR. Unvaped JUUL "Menthol" pods contained ∼621.3 mg/mL of G, ∼264.9 mg/mL of PG, ∼59.3 mg/mL of nicotine, ∼13.3 mg/mL of menthol, and ∼0.1 mg/mL of the coolant WS-23. Eleven experienced male EC users (aged 21-26) provided exhaled aerosol and residue samples before and after vaping JUUL pods. Participants vaped ad libitum for 20 min, while their average puff count (22 ± 6.4) and puff duration (4.4 ± 2.0) were recorded. The transfer efficiency of nicotine, menthol, and WS-23 from the pod fluid into the aerosol varied with each chemical and was generally similar across flow rates (9-47 mL/s). At 21 mL/s, the average mass of each chemical retained by the participants who vaped 20 min was 53.2 ± 40.3 mg for G, 18.9 ± 14.3 mg for PG, 3.3 ± 2.7 mg for nicotine, and 0.5 ± 0.4 mg for menthol, with retention deduced to be ∼90-100% for each chemical. There was a significant positive relationship between the number of symptoms during vaping and total chemical mass retained. ECEAR accumulated on enclosed surfaces where it could contribute to passive exposure. These data will be valuable to researchers studying human exposure to EC aerosols and agencies that regulate EC products.

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.

Chemical Elements, Flavor Chemicals, and Nicotine in Unused and Used Electronic Cigarettes Aged 5-10 Years and Effects of pH.

The concentrations of elements/metals, nicotine, flavor chemicals and acids were compared in the e-liquids of unused and used first-generation electronic cigarettes (ECs) that were stored for 5-10 years. Metal analysis was performed using inductively coupled plasma optical emission spectroscopy; nicotine and flavor chemical analyses were performed using gas chromatography/mass spectroscopy. Of the 22 elements analyzed, 10 (aluminum, chromium, copper, iron, lead, nickel, selenium, silicon, tin, zinc) were often found in the e-liquids. Five elements had the highest average concentrations: copper (1161.6 mg/L), zinc (295.8 mg/L), tin (287.6 mg/L), nickel (71.1 mg/L), and lead (50.3 mg/L). Nicotine concentrations were always lower than label concentrations indicated. Of the 181 flavor chemicals analyzed, 11 were detected in at least one sample, with hydroxyacetone being present in all samples. In used products, some flavor chemicals appeared to be by-products of heating. E-liquids with the highest concentrations of acids and the lowest pH levels also had the highest concentrations of elements/metals. Metal concentrations in e-liquids increased after use in some products, and some metal concentrations, such as nickel, were high enough to be a health concern. Leachates from discarded ECs could contribute toxic metals/chemicals to the environment, supporting the need for better regulation of atomizer design, composition, and disposal.

Ethyl maltol, vanillin, corylone and other conventional confectionery-related flavour chemicals dominate in some e-cigarette liquids labelled 'tobacco' flavoured.

BACKGROUND: The increased popularity of electronic cigarettes (e-cigarettes) has been linked to the abundance of flavoured products that are attractive to adolescents and young adults. In the last decade, e-cigarette designs have evolved through four generations that include modifications in battery power, e-cigarette liquid (e-liquid) reservoirs and atomiser units. E-liquids have likewise evolved in terms of solvent use/ratios, concentration and number of flavour chemicals, use of nicotine salts and acids, the recent increased use of synthetic cooling agents and the introduction of synthetic nicotine. Our current objective was to evaluate and compare the evolving composition of tobacco-flavoured e-liquids over the last 10 years. METHODS: Our extensive database of flavour chemicals in e-liquids was used to identify trends and changes in flavour chemical composition and concentrations. RESULTS: Tobacco-flavoured products purchased in 2010 and 2011 generally had very few flavour chemicals, and their concentrations were generally very low. In tobacco-flavoured refill fluids purchased in 2019 and Puff Bar Tobacco e-cigarettes, the total number and concentration of flavour chemicals were higher than expected. Products with total flavour chemicals >10 mg/mL contained one to five dominant flavour chemicals (>1 mg/mL). The most frequently used flavour chemicals in tobacco e-liquids were fruity and caramellic. CONCLUSIONS: There is a need for continuous surveillance of e-liquids, which are evolving in often subtle and harmful ways. Chemical constituents of tobacco flavours should be monitored as they clearly can be doctored by manufacturers to have a taste that would appeal to young users.

Disposable Puff Bar Electronic Cigarettes: Chemical Composition and Toxicity of E-liquids and a Synthetic Coolant.

The popularity of disposable fourth-generation electronic cigarettes (ECs) among young adults and adolescents has been increasing since the ban on flavored cartridge EC products such as JUUL. Although the constituents and toxicity of some cartridge-based fourth-generation ECs, such as JUUL, have been studied, limited data exist for other disposable ECs such as Puff. The purpose of this study was to determine flavor chemicals, synthetic coolants, and nicotine concentrations in 16 disposable Puff devices, evaluate the cytotoxicity of the different flavors from the Puff brand using in vitro assays, and investigate the health risks of synthetic coolants in EC products. Gas chromatography/mass spectrometry was used to identify and quantify chemicals in Puff EC fluids. One hundred and twenty-six flavor chemicals were identified in Puff fluids, and 16 were >1 mg/mL. WS-23 (2-isopropyl-N,2,3-trimethylbutyramide) was present in all products, and concentrations ranged from 0.8 to 45.1 mg/mL. WS-3 (N-ethyl-p-menthane-3-carboxamide) concentrations ranged from 1.5 to 16.4 mg/mL in 6/16 products. Nicotine concentrations ranged from 40.6 to 52.4 (average 44.8 mg/mL). All unvaped fluids were cytotoxic at dilutions between 0.1 and 10% in the MTT and neutral red uptake assays when tested with BEAS-2B lung epithelial cells. The cytotoxicity of Puff fluids was highly correlated with total chemical concentrations, nicotine, WS-23, both synthetic coolants, and synthetic coolants plus ethyl maltol. Lower concentrations of WS-23 than those in the fluids adversely affected cell growth and morphology. Concentrations of synthetic coolants exceeded levels used in consumer products. The margin of exposure data showed that WS-3 and WS-23 concentrations were high enough in Puff products to present a health hazard. Our study demonstrates that disposable Puff ECs have high levels of cytotoxic chemicals. The data support the regulation of flavor chemicals and synthetic coolants in ECs to limit potentially harmful health effects.

Flavour chemicals, synthetic coolants and pulegone in popular mint-flavoured and menthol-flavoured e-cigarettes.

BACKGROUND: The Food and Drug Administration (FDA) has recently banned flavours from pod-style electronic cigarettes (e-cigarettes), except for menthol and tobacco. JUUL customers have quickly discovered that flavoured disposable e-cigarettes from other manufacturers, such as Puff, are readily available. Our goal was to compare flavour chemicals, synthetic coolants and pulegone in mint-flavoured/menthol-flavoured e-cigarettes from JUUL and Puff, evaluate the cytotoxicity of the coolants and perform a cancer risk assessment for pulegone, which is present in both JUUL pods and disposable Puff products. METHODS: Identification and quantification of chemicals were performed using gas chromatography/mass spectrometry. Cytotoxicity of the coolants was evaluated with BEAS-2B cells using the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cancer risk of pulegone was calculated using the margin of exposure (MOE). RESULTS: Menthol was the dominant flavour chemical (>1 mg/mL) in all products from both manufacturers. Minor flavour chemicals (<1 mg/mL) differed in the JUUL and Puff fluids and may produce flavour accents. The concentrations of WS-3 and WS-23 were higher in Puff than in JUUL. WS-23 was cytotoxic in the MTT assay at concentrations 90 times lower than concentrations in Puff fluids. The risk of cancer (MOE<10 000) was greater for mint than for menthol products and greater for Puff than for JUUL. CONCLUSIONS: Switching from flavoured JUUL to Puff e-cigarettes may expose users to increased harm due to the higher levels of WS-23 and pulegone in Puff products. Cancer risk may be reduced in e-cigarettes by using pure menthol rather than mint oils to produce minty-flavoured e-cigarette products.

'Menthol-Plus': a major category of cigarette found among 'concept' descriptor cigarettes from Mexico.

BACKGROUND: Tobacco companies are offering cigarettes with 'concept' descriptor names that suggest sensation and/or flavour properties (eg, Marlboro 'Velvet Fusion'). Little has been known about the identities and levels of flavour chemicals in such cigarettes. METHODS: Thirty-three filter cigarette variants from 27 packs (including two sampler packs with four variations each) from Canada and Mexico were analysed (rod + filter) for 177 flavour chemicals plus triacetin, a filter plasticiser and possible flavourant. Five brands of US mentholated filter cigarettes were also analysed. RESULTS: Twenty-seven of the 33 cigarettes (all were Mexican variants) were categorised as 'menthol-plus': significant menthol (3.0-11.9 mg/cigarette), plus varying amounts (0.32-3.4 mg/cigarette) of total other flavour chemicals (TOFCs) (excludes triacetin). For 10 of the 27, TOFCs >1.0 mg/cigarette. For 7 of the 27, the TOFCs profile was categorised as containing total fruit flavour compounds (TFFCs) >1.0 mg/cigarette. One Mexican variant was categorised as 'menthol-only' (TOFCs ≤0.15 mg/cigarette). All menthol-plus and menthol-only cigarettes contained one or two optional-crush capsules in their filters (crushed prior to analysis). All five Canadian brand variants were 'non-flavoured'. All five US brand variants were 'menthol-only'. CONCLUSIONS: All but one of the 'concept' descriptor cigarettes from Mexico were 'menthol-plus'. While the Canadian cigarettes complied with Canada's flavour chemical ban, concept descriptors on the packs may increase appeal. Given the scale of the problem posed by menthol alone, health officials seeking to decrease the appeal of smoked tobacco should examine the extent to which 'concept descriptor' cigarettes using 'menthol-plus' flavour profiling together with artful descriptors are furthering the problem of smoked tobacco.

Tracing the movement of electronic cigarette flavor chemicals and nicotine from refill fluids to aerosol, lungs, exhale, and the environment.

BACKGROUND: Given the high concentrations of nicotine and flavor chemicals in EC (electronic cigarette) fluids, it is important to determine how efficiently they transfer to aerosols, how well they are retained by users (exposure), and if they are exhaled into the environment where they settle of surfaces forming ECEAR (EC exhaled aerosol residue). OBJECTIVES: To quantify the flavor chemicals and nicotine in refill fluids, inhaled aerosols, and exhaled aerosols. Then deduce their retention and contribution to ECEAR. METHODS: Flavor chemicals and nicotine were identified and quantified by GC-MS in two refill fluids, smoking machine-generated aerosols, and aerosols exhaled by 10 human participants (average age 21; 7 males). Machine generated aerosols were made with varying puff durations and two wattages (40 and 80). Participants generated exhale ad libitum; their exhale was quantified, and chemical retention and contribution to ECEAR was modeled. RESULTS: "Dewberry Cream" had five dominant (≥1 mg/mL) flavor chemicals (maltol, ethyl maltol, vanillin, ethyl vanillin, furaneol), while "Cinnamon Roll" had one (cinnamaldehyde). Nicotine transferred well to aerosols irrespective of topography; however, transfer efficiencies of flavor chemicals depended on the chemical, puff volume, puff duration, pump head, and EC power. Participants could be classified as "mouth inhalers" or "lung inhalers" based on their exhale of flavor chemicals and nicotine and retention. Lung inhalers had high retention and exhaled low concentrations of EC chemicals. Only mouth inhalers exhaled sufficient concentrations of flavor chemicals/nicotine to contribute to chemical deposition on environmental surfaces (ECEAR). CONCLUSION: These data help distinguish two types of EC users, add to our knowledge of chemical exposure during vaping, and provide information useful in regulating EC use.

Measurement of the Free-Base Nicotine Fraction (αfb) in Electronic Cigarette Liquids by Headspace Solid-Phase Microextraction.

A method for determining the fraction of free-base nicotine (αfb) in electronic cigarette liquids ("e-liquids") based on headspace solid-phase microextraction (h-SPME) is described. The free-base concentration ce,fb = αfbce,T, where ce,T is the total (free-base + protonated) nicotine in the liquid. For gas/liquid equilibrium of the volatile free-base form, the headspace nicotine concentration is proportional to ce,fb and thus also to αfb. Headspace nicotine is proportionally absorbed with an SPME fiber. The fiber is thermally desorbed in the heated inlet of a gas chromatograph coupled to a mass spectrometer: the desorbed nicotine is measured by gas chromatography-mass spectrometry. For a second h-SPME measurement, an adequate base is added to the sample vial to convert essentially all protonated nicotine to the free-base form (αfb → 1.0). The ratio of the first h-SPME measurement to the second h-SPME measurement gives αfb in the initial sample. Using gaseous ammonia as the added base, the method was (1) verified using lab-prepared e-liquid solutions with known αfb values and (2) used to determine the αfb values for 18 commercial e-liquids. The measured αfb values ranged from 0.0 to 1.0. Increasing measurement error with decreasing αfb caused modestly lower method precision at small αfb. Adding a liquid organic base may be more convenient than adding gaseous ammonia: one of the samples was examined using triethylamine as the added base; the measurements agreed well (with ammonia, 0.27 ± 0.01; with triethylamine, 0.26 ± 0.04). Other workers have proposed examining the nicotine protonation state in e-liquids using three steps: (1) 1:10 dilution with CO2-free water; (2) measurement of pH; and (3) calculation of the resulting values for αfb,w,1:10, the free-base fraction in the diluted mostly aqueous phase. As expected and verified here, because of the generally greater abilities of organic acids to protonate nicotine in water versus in an e-liquid phase, αfb,w,1:10 values can be significantly less than actual e-liquid αfb values when αfb is not close to either 0 or 1.

E-cigarette fluids and aerosol residues cause oxidative stress and an inflammatory response in human keratinocytes and 3D skin models.

Our goal was to evaluate the effects of EC refill fluids and EC exhaled aerosol residue (ECEAR) on cultured human keratinocytes and MatTek EpiDerm™, a 3D air liquid interface human skin model. Quantification of flavor chemicals and nicotine in Dewberry Cream and Churrios refill fluids was done using GC-MS. The dominant flavor chemicals were maltol, ethyl maltol, vanillin, ethyl vanillin, benzyl alcohol, and furaneol. Cytotoxicity was determined with the MTT and LDH assays, and inflammatory markers were quantified with ELISAs. Churrios was cytotoxic to keratinocytes in the MTT assay, and both fluids induced ROS production in the medium (ROS-Glo™) and in cells (CellROX). Exposure of EpiDerm™ to relevant concentrations of Dewberry Cream and Churrios for 4 or 24 h caused secretion of inflammatory markers (IL-1α, IL-6, and MMP-9), without altering EpiDerm™ histology. Lab made fluids with propylene glycol (PG) or PG plus a flavor chemical did not produce cytotoxic effects, but increased secretion of IL-1α and MMP-9, which was attributed to PG. ECEAR derived from Dewberry Cream and Churrios did not produce cytotoxicity with Epiderm™, but Churrios ECEAR induced IL-1α secretion. These data support the conclusion that EC chemicals can cause oxidative damage and inflammation to human skin.

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.

The influence of terpenes on the release of volatile organic compounds and active ingredients to cannabis vaping aerosols.

Dabbing and vaping cannabis extracts have gained large popularity in the United States as alternatives to cannabis smoking, but diversity in both available products and consumption habits make it difficult to assess consumer exposure to psychoactive ingredients and potentially harmful components. This work studies the how relative ratios of the two primary components of cannabis extracts, Δ9-tetrahydrocannabinol (THC) and terpenes, affect dosage of these and exposure to harmful or potentially harmful components (HPHCs). THC contains a monoterpene moiety and has been previously shown to emit similar volatile degradation products to terpenes when vaporized. Herein, the major thermal degradation mechanisms for THC and ß-myrcene are elucidated via analysis of their aerosol gas phase products using automated thermal desorption-gas chromatography-mass spectrometry with the aid of isotopic labelling and chemical mechanism modelling. Four abundant products - isoprene, 2-methyl-2-butene, 3-methylcrotonaldehyde, and 3-methyl-1-butene - are shown to derive from a common radical intermediate for both THC and ß-myrcene and these products comprise 18-30% of the aerosol gas phase. The relative levels of these four products are highly correlated with applied power to the e-cigarette, which indicates formation of these products is temperature dependent. Vaping THC-ß-myrcene mixtures with increasing % mass of ß-myrcene is correlated with less degradation of the starting material and a product distribution suggestive of a lower aerosolization temperature. By contrast, dabbing THC-ß-myrcene mixtures with increasing % mass of ß-myrcene is associated with higher levels of HPHCs, and isotopic labelling showed this is due to increased reactivity of ß-myrcene relative to THC.

Electronic Cigarette Refill Fluids Sold Worldwide: Flavor Chemical Composition, Toxicity, and Hazard Analysis.

Flavor chemicals in electronic cigarette (EC) fluids, which may negatively impact human health, have been studied in a limited number of countries/locations. To gain an understanding of how the composition and concentrations of flavor chemicals in ECs are influenced by product sale location, we evaluated refill fluids manufactured by one company (Ritchy LTD) and purchased worldwide. Flavor chemicals were identified and quantified using gas chromatography/mass spectrometry (GC/MS). We then screened the fluids for their effects on cytotoxicity (MTT assay) and proliferation (live-cell imaging) and tested authentic standards of specific flavor chemicals to identify those that were cytotoxic at concentrations found in refill fluids. A total of 126 flavor chemicals were detected in 103 bottles of refill fluid, and their number per/bottle ranged from 1-50 based on our target list. Two products had none of the flavor chemicals on our target list, nor did they have any nontargeted flavor chemicals. A total of 28 flavor chemicals were present at concentrations ≥1 mg/mL in at least one product, and 6 of these were present at concentrations ≥10 mg/mL. The total flavor chemical concentration was ≥1 mg/mL in 70% of the refill fluids and ≥10 mg/mL in 26%. For sub-brand duplicate bottles purchased in different countries, flavor chemical concentrations were similar and induced similar responses in the in vitro assays (cytotoxicity and cell growth inhibition). The levels of furaneol, benzyl alcohol, ethyl maltol, ethyl vanillin, corylone, and vanillin were significantly correlated with cytotoxicity. The margin of exposure calculations showed that pulegone and estragole levels were high enough in some products to present a nontrivial calculated risk for cancer. Flavor chemical concentrations in refill fluids often exceeded concentrations permitted in other consumer products. These data support the regulation of flavor chemicals in EC products to reduce their potential for producing both cancer and noncancer toxicological effects.

Menthol in electronic cigarettes: A contributor to respiratory disease?

Menthol is widely used in tobacco products. This study compared the effects of menthol on human bronchial epithelium using submerged cultures, a VITROCELL® cloud chamber that provides air liquid interface (ALI) exposure without solvents or heating, and a Cultex ALI system that delivers aerosol equivalent to that inhaled during vaping. In submerged culture, menthol significantly increased calcium influx and mitochondrial reactive oxygen species (ROS) via the TRPM8 receptor, responses that were inhibited by a TRPM8 antagonist. VITROCELL® cloud chamber exposure of BEAS-2B monolayers increased mitochondrial protein oxidation, expression of the antioxidant enzyme SOD2, activation of NF-κB, and secretion of inflammatory cytokines (IL-6 and IL-8). Proteomics data collected following ALI exposure of 3D EpiAirway tissue in the Cultex showed upregulation of NRF-2-mediated oxidative stress, oxidative phosphorylation, and IL-8 signaling. Across the three platforms, menthol adversely effected human bronchial epithelium in a manner that could lead to respiratory disease.

Daily stream samples reveal highly complex pesticide occurrence and potential toxicity to aquatic life.

Transient, acutely toxic concentrations of pesticides in streams can go undetected by fixed-interval sampling programs. Here we compare temporal patterns in occurrence of current-use pesticides in daily composite samples to those in weekly composite and weekly discrete samples of surface water from 14 small stream sites. Samples were collected over 10-14 weeks at 7 stream sites in each of the Midwestern and Southeastern United States. Samples were analyzed for over 200 pesticides and degradates by direct aqueous injection liquid chromatography with tandem mass spectrometry. Nearly 2 and 3 times as many unique pesticides were detected in daily samples as in weekly composite and weekly discrete samples, respectively. Based on exceedances of acute-invertebrate benchmarks (AIB) and(or) a Pesticide Toxicity Index (PTI) >1, potential acute-invertebrate toxicity was predicted at 11 of 14 sites from the results for daily composite samples, but was predicted for only 3 sites from weekly composites and for no sites from weekly discrete samples. Insecticides were responsible for most of the potential invertebrate toxicity, occurred transiently, and frequently were missed by the weekly discrete and composite samples. The number of days with benthic-invertebrate PTI ≥0.1 in daily composite samples was inversely related to Ephemeroptera, Plecoptera, and Trichoptera (EPT) richness at the sites. The results of the study indicate that short-term, potentially toxic peaks in pesticides frequently are missed by weekly discrete sampling, and that such peaks may contribute to degradation of invertebrate community condition in small streams. Weekly composite samples underestimated maximum concentrations and potential acute-invertebrate toxicity, but to a lesser degree than weekly discrete samples, and provided a reasonable approximation of the 90th percentile total concentrations of herbicides, insecticides, and fungicides, suggesting that weekly composite sampling may be a compromise between assessment needs and cost.