Stephen B. Stanfill

Application of comprehensive two-dimensional gas chromatography (GC×GC) to the qualitative analysis of essential oils.

This paper investigates the separation of moderately complex samples by comprehensive two-dimensional gas chromatography (GC x GC). The analysis of peppermint (Mentha piperita) and spearmint (Mentha spicata) essential oil components, including acetates, alcohols, furans, ketones, sesquiterpenes, and terpenes, was achieved by one-dimensional gas chromatography with quadrupole mass spectrometry detection (GC/MSD) and GC x GC with flame ionization detection. Peppermint essential oil was found to contain 89 identifiable peaks by GC x GC compared to 30 peaks in the GC/MSD chromatogram. Likewise, 68 peaks were found in the GC x GC chromatogram of spearmint (compared to 28 in GC/MSD). Plots of the first dimension versus second dimension retention times provided a fingerprint of the two essential oils, which revealed 52 similar compounds between the two essential oils as opposed to 18 matches by ID GC.

Global surveillance of oral tobacco products: total nicotine, unionised nicotine and tobacco-specific N-nitrosamines

Objective Oral tobacco products contain nicotine and carcinogenic tobacco-specific N-nitrosamines (TSNAs) that can be absorbed through the oral mucosa. The aim of this study was to determine typical pH ranges and concentrations of total nicotine, unionised nicotine (the most readily absorbed form) and five TSNAs in selected oral tobacco products distributed globally. Methods A total of 53 oral tobacco products from 5 World Health Organisation (WHO) regions were analysed for total nicotine and TSNAs, including 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), using gas chromatography or liquid chromatography with mass spectrometric detection. Unionised nicotine concentrations were calculated using product pH and total nicotine concentrations. Fourier transform infrared spectroscopy was used to help categorise or characterise some products. Results Total nicotine content varied from 0.16 to 34.1 mg/g product, whereas, the calculated unionised nicotine ranged from 0.05 to 31.0 mg/g product; a 620-fold range of variation. Products ranged from pH 5.2 to 10.1, which translates to 0.2% to 99.1% of nicotine being in the unionised form. Some products have very high pH and correspondingly high unionised nicotine (eg, gul powder, chimó, toombak) and/or high TSNA (eg, toombak, zarda, khaini) concentrations. The concentrations of TSNAs spanned five orders of magnitude with concentrations of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) ranging from 4.5 to 516 000 ng/g product. Conclusions These data have important implications for risk assessment because they show that very different exposure risks may be posed through the use of these chemically diverse oral tobacco products. Because of the wide chemical variation, oral tobacco products should not be categorised together when considering the public health implications of their use.

Surveillance of moist snuff: total nicotine, moisture, pH, un-ionized nicotine, and tobacco-specific nitrosamines

In 2005, approximately 2.3% of U.S. adults used smokeless tobacco. Moist snuff leads all types of smokeless tobacco in revenues and marketing expenditures. The U.S. Surgeon General has concluded that smokeless tobacco use can lead to nicotine addiction. The National Toxicology Program of the National Institutes of Health has classified smokeless tobacco as a human carcinogen. Tobacco-specific nitrosamines (TSNAs) are potent carcinogens in smokeless tobacco products, and the pH of the product influences the content of un-ionized nicotine which is the form of nicotine most rapidly absorbed in the mouth. The Centers for Disease Control and Prevention analyzed 40 top-selling brands of moist snuff to measure nicotine, moisture, pH, un-ionized nicotine, and TSNAs, including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). The study findings indicate that moist snuff brands varied widely in content of rapidly absorbed, addictive un-ionized nicotine (500-fold range) and of carcinogenic TSNAs (18-fold range). Product characteristics such as packaging and moisture content appeared to be correlated with concentrations of un-ionized nicotine, and flavor characteristics of low-priced brands may correlate with TSNA concentrations. These findings warrant further study in light of (a) the marketing of smokeless tobacco for use in places where smoking is prohibited, (b) the promotion of smokeless tobacco as a harm-reduction product, and (c) the ever-expanding number of highly flavored smokeless varieties brought to the market.

Chemical Composition and Evaluation of Nicotine, Tobacco Alkaloids, pH, and Selected Flavors in E-Cigarette Cartridges and Refill Solutions

INTRODUCTION Electronic cigarette (e-cigarette) use is increasing dramatically in developed countries, but little is known about these rapidly evolving products. This study analyzed and evaluated the chemical composition including nicotine, tobacco alkaloids, pH, and flavors in 36 e-liquids brands from 4 manufacturers. METHODS We determined the concentrations of nicotine, alkaloids, and select flavors and measured pH in solutions used in e-cigarettes. E-cigarette products were chosen based upon favorable consumer approval ratings from online review websites. Quantitative analyses were performed using strict quality assurance/quality control validated methods previously established by our lab for the measurement of nicotine, alkaloids, pH, and flavors. RESULTS Three-quarters of the products contained lower measured nicotine levels than the stated label values (6%-42% by concentration). The pH for e-liquids ranged from 5.1-9.1. Minor tobacco alkaloids were found in all samples containing nicotine, and their relative concentrations varied widely among manufacturers. A number of common flavor compounds were analyzed in all e-liquids. CONCLUSIONS Free nicotine levels calculated from the measurement of pH correlated with total nicotine content. The direct correlation between the total nicotine concentration and pH suggests that the alkalinity of nicotine drives the pH of e-cigarette solutions. A higher percentage of nicotine exists in the more absorbable free form as total nicotine concentration increases. A number of products contained tobacco alkaloids at concentrations that exceed U.S. pharmacopeia limits for impurities in nicotine used in pharmaceutical and food products.

Rapid quantitation of cyanide in whole blood by automated headspace gas chromatography

Cyanide (CN), a chemical asphyxiant, is a rapidly acting and powerful poison. We have developed a sensitive, rapid, simple, and fully automated method for measuring CN in whole blood. The assay is based on the use of gas chromatography (GC) with nitrogen-phosphorus detection and acetonitrile as an internal reference. Following the automated addition of phosphoric acid to the blood sample, the released hydrogen cyanide is analyzed using a fully automated headspace GC system. The assay, validated on human blood samples spiked with potassium cyanide and on clinical samples from fire victims who had smoke inhalation injury, can detect CN at a wide range of concentrations (30-6000 microg/l) in about 17 min (including incubation and GC run time, and <2 min for manual sample preparation). This automated, high-throughput, simple, and sensitive method is suitable for the rapid diagnosis of CN in clinical and forensic specimens.

Concentrations of nine alkenylbenzenes, coumarin, piperonal and pulegone in Indian bidi cigarette tobacco.

Indian-made bidi cigarettes sold in the United States are available in a variety of exotic (e.g. clove, mango) and candy-like (e.g. chocolate, raspberry) flavors. Because certain tobacco flavorings contain alkenylbenzenes and other toxic or carcinogenic chemicals, we measured the concentration of flavor-related compounds in bidi tobacco using a previously developed method. Twenty-three brands of bidis were sampled using automated headspace solid-phase microextraction and subsequently analyzed for 12 compounds by gas chromatography-mass spectrometry. Two alkenylbenzene compounds, trans-anethole and eugenol, were found in greater than 90% of the brands analyzed. Methyleugenol, pulegone and estragole were each detected in 30% or more of the brands, whereas safrole and elemicin were not detected in any of the brands. The flavor-related compounds with the highest tobacco concentrations were eugenol (12,000 microg/g tobacco) and trans-anethole (2200 microg/g tobacco). The highest eugenol and trans-anethole concentrations found in bidi tobacco were about 70,000 and 7500 times greater, respectively, than the highest levels previously found in US cigarette brands. Measurement of these compounds is crucial to evaluation of potential risks associated with inhaling highly concentrated flavor-related compounds from bidis or other tobacco products.

Unintentional Child Poisonings Through Ingestion of Conventional and Novel Tobacco Products

OBJECTIVE: This study examines child poisonings resulting from ingestion of tobacco products throughout the nation and assesses the potential toxicity of novel smokeless tobacco products, which are of concern with their discreet form, candy-like appearance, and added flavorings that may be attractive to young children. METHODS: Data representing all single-substance, accidental poisonings resulting from ingestion of tobacco products by children <6 years of age, reported to poison control centers, were examined. Age association with ingestion of smokeless tobacco versus other tobacco products was tested through logistic regression. Total nicotine content, pH, and un-ionized nicotine level were determined, and the latter was compared with values for moist snuff and cigarettes. RESULTS: A total of 13705 tobacco product ingestion cases were reported, >70% of which involved infants <1 year of age. Smokeless tobacco products were the second most common tobacco products ingested by children, after cigarettes, and represented an increasing proportion of tobacco ingestions with each year of age from 0 to 5 years (odds ratio: 1.94 [95% confidence interval: 1.86–2.03]). A novel, dissolvable, smokeless tobacco product with discreet form, candy-like appearance, and added flavorings was found to contain an average of 0.83 mg of nicotine per pellet, with an average pH of 7.9, which resulted in an average of 42% of the nicotine in the un-ionized form. CONCLUSION: In light of the novelty and potential harm of dissolvable nicotine products, public health authorities are advised to study these products to determine the appropriate regulatory approach.

Solid phase microextraction of alkenylbenzenes and other flavor-related compounds from tobacco for analysis by selected ion monitoring gas chromatography–mass spectrometry

Some constituents found in natural flavorings are known to exhibit toxic properties. We developed a rapid method for quantifying 12 flavor-related compounds in cigarette tobacco using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. Using selected ion monitoring, we quantified and positively identified coumarin; pulegone; piperonal and nine alkenylbenzenes, including trans-anethole, safrole, methyleugenol and myristicin in one or more brands of cigarettes. In 62% of 68 brands analyzed, we detected one or more of the flavor-related compounds ranging from 0.0018 to 43 microg/g. Toxic properties of these flavor-related compounds may constitute an additional health risk related to cigarette smoking.

Chemical characterization of domestic oral tobacco products: Total nicotine, pH, unprotonated nicotine and tobacco-specific N-nitrosamines

In the United States, moist snuff has been studied more widely than other distinct categories of oral tobacco. In this study, we measured pH, moisture, nicotine (total and unprotonated), and tobacco-specific N-nitrosamines (TSNAs) for other established (twist, loose leaf, plug, and dry snuff without pouch) and emerging oral tobacco products (dry snuff pouch, US-made snus, and dissolvable tobacco). Among the seven product categories, product pH ranged from 4.7 to 7.9, and total nicotine concentration spanned from 3.9 to 40.1 mg/g. The most readily absorbable form of nicotine (unprotonated nicotine) varied more than 350-fold, ranging from 0.01 to 3.7 mg/g. While the highest total nicotine concentrations were observed in twist products, snus and dissolvable tobacco had the highest unprotonated nicotine levels. Among all products, total TSNA concentrations ranged from 313 to 76,500 ng/g with dry snuff having the highest total TSNA concentrations. This study demonstrates the diversity among oral tobacco products and highlights the potential of these products to deliver a wide range of nicotine and carcinogenic TSNAs. Characterizing the chemical content of these products may be helpful in further understanding the risk of marketing these products to oral tobacco users and smokers as an alternative and discrete form of tobacco.

Automated determination of seven phenolic compounds in mainstream tobacco smoke

Exposure to hydroxyl-substituted arenes, commonly referred to as phenols or phenolic compounds, can have serious health consequences. Select phenols present in tobacco smoke are cardiovascular toxins, act as tumor co-promoters and show genotoxic activity. To examine the mainstream smoke levels of these compounds, we developed and applied a method for quantitative analysis of seven phenols (phenol, o-cresol, m-cresol, p-cresol, catechol, resorcinol, and hydroquinone) in mainstream smoke. Total mainstream smoke particulate matter was collected on a Cambridge filter pad and spiked with an isotopically labeled internal standard solution. This pad underwent an automated phenol derivatization procedure to increase analyte volatility and enhance detection. Following the derivatization step, phenols from the particulate matter were sampled using solid-phase microextraction with subsequent gas chromatography/mass spectrometric detection. Sensitivity, selectivity, accuracy, and reproducibility were more than adequate for routine detection of phenols in mainstream smoke. Detection limits ranged from 0.04-0.57 microg, with a quantification range of 0.1-710 microg. Higher sensitivity and sample throughput were achieved compared with previously described methods. Mainstream smoke from 28 brands of domestic commercial cigarettes was evaluated to assess typical levels, and reference cigarettes containing single tobacco blends were examined to ascertain the phenolic profile from different types of tobaccos. As expected under machine smoking conditions using the Federal Trade Commission parameters, full-flavored cigarettes deliver more phenols than the light varieties, followed by the ultra light varieties. Differences were seen in relative levels of phenolic compounds in the mainstream smoke from unfiltered cigarettes made with a single type of tobacco.