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Introduction. Popularity of electronic cigarettes (e-cigarettes) has increased dramatically in recent years, especially among adolescents. The most recent data from the National Institute on Drug Abuse (NIDA) cites that >16% of 12th graders have tried e-cigarettes, and >30% of those individuals will start smoking within 6 months1. E-cigarettes are available in a variety of ‘strengths’ indicating the labeled nicotine concentration in the product. In this project, we sought to investigate the accuracy of nicotine labeling found in some commercially available e-liquids. As such, we developed and validated a liquid chromatography-mass spectrometry (LC-MS) assay to determine the nicotine concentration in these products. Methods. A literature search revealed that the two most commonly used chromatographic approaches for nicotine are hydrophilic interaction liquid chromatography (HILIC) and reversed phase chromatography (RP). Both options were evaluated, and nicotine peak quality and reproducibility were assessed. Mass spectrometric conditions for positive electrospray (+ESI) ionization were optimized, including collision energy and ion accumulation time. The optimized method included a gradient separation using a UCT C18 column (2.1 x 100 mm; 1.8 micron) with acetonitrile as the organic phase and 0.1% formic acid in water as the aqueous phase. Nicotine stock solutions were prepared in 100% ethanol and diluted in acetonitrile to achieve calibration concentrations (5 – 75 micrograms/mL). Deuterium labeled nicotine (d4) was used as the internal standard at a concentration of 10 micrograms/mL. The method was evaluated for precision, reflected by percent relative standard deviation (%RSD) and accuracy, or percent error, at each concentration for three days. The method was applied to the assessment of nicotine concentration in samples of e-liquids labeled as 3 mg/mL nicotine. Results. Reversed phase chromatography outperformed HILIC separation for nicotine under the conditions tested. The final RP-LC-MS/MS method involves direct monitoring of m/z 163.1219 for quantification of nicotine (167.1219 for d4-nicotine). The method exhibits < 15% RSD and < 15% error for all concentrations in the calibration range (< 20% at the lower limit of quantification). The developed method allows for rapid throughput, with a run time of 5 minutes. The lower limit of detection was determined to be 1 microgram/mL. Of the e-liquids evaluated, variations of up to 37% from the labeled amount of 3 mg/mL were detected. Additionally, a product labeled ‘zero nicotine’ contained no detectable nicotine. Conclusions. A fast, accurate, and reproducible LC-MS/MS assay has been developed and validated for the determination of nicotine in e-cigarette liquids. This method was applied to the evaluation of e-liquids, which showed significant variability in nicotine content from the labeled amount. 1 https://www.drugabuse.gov/related-topics/trends-statistics/infographics/teens-e-cigarettes

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Smoking combustible cigarettes is the major cause of disease and death among adults living in the United States (U.S.). In fact, smoking combustible cigarettes causes nearly half a million premature deaths among U.S. adults every year.1–4 It is estimated that over 14% (equating to 34 million persons) of U.S. adults smoke cigarettes currently.2,5 This is a substantial decrease from 1965 when it was estimated that more than 42% of U.S. adults smoked cigarettes.1 This is partly attributed to the well-established evidence that smoking cigarettes causes harm to almost every human organ system5 and is associated with an elevated risk of developing cancer6, cardiovascular disease 7, pulmonary disease and respiratory illnesses.8–10 Despite the well-established health consequences of smoking cigarettes, millions of people are still smoking, which alone suggests that nicotine (the primary constituent of cigarettes) is highly addictive.3 Thankfully, smoking cessation by means of pharmacological treatments has been shown to help smokers overcome nicotine addiction. A review of the research on the efficacy of varenicline (Chantix) and Nicotine Replacement Therapy (NRT), two of the most commonly used smoking cessation treatments, reveals that both treatments increase long-term smoking abstinence rates with odds ratios of 3.85 for varenicline and 1.74 for NRT when compared to placebo.11–14 Even more, both drugs appear to be generally well-tolerated, with no known life-threatening side effects when compared to placebo. Research shows that the most common side effects for varenicline are nausea, insomnia, gastrointestinal effects, headache and abnormal dreams.11,15 The most common side effects for NRT appear to be skin irritation, insomnia, headache, nausea/vomiting and cough.11,15 Ultimately, both varenicline and NRT appear to be strong options for achieving smoking abstinence both with respect to overall efficacy and tolerability.

E-cigarettes have recently become increasingly popular, especially amongst middle and high school students. Although they are marketed as safer alternatives to tobacco cigarettes, they produce toxic metals and carcinogenic nitrosamines. This thesis studies the effects of e-cigarette aerosol on the growth and proliferation of oral epithelial cells because the consequences of vaping, including a potential risk for aberrant growth leading to cancer, are not well understood. Cells were grown in matrigel, causing the formation of three-dimensional spheroids modeling the physiological architecture of the oral epithelium. Those spheroids were chronically exposed to vape with different treatment conditions to study the functional biological effects of the presence of nicotine, dosage, and different types of exposure. The diameter of the spheroids was measured throughout the process as an indicator of cell growth. It showed that the vape exposure, especially nicotine-rich aerosol, induces an increase in spheroid diameter in a dose-dependent manner. The increased cell growth is supported by enhanced metabolic activity as well as increased aldehyde dehydrogenase activity, a marker of stemness prominently upregulated in cancer stem cells. Protein was extracted at the end for protein expression analysis through Western blotting and the identification of the activation of survival signaling pathways and stem cell markers. Lastly, spheroids were co-cultured with Strep. mutans, a cariogenic bacterial resident in the oral cavity, and acutely exposed to vape. Co-culture with S. mutans did not significantly affect spheroid growth under the current experimental conditions or significantly change the expression patterns of proliferation and tumor initiation proteins. Future research will include tumorigenic assays and investigate how vape may induce carcinogenesis of the oral epithelium.

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