Improving Wastewater-Based Tobacco Use Estimates Using Anabasine.

In wastewater-based epidemiology (WBE), nicotine metabolites have been used as biomarkers for monitoring tobacco use. Recently, the minor tobacco alkaloids anabasine and anatabine have been suggested as more specific biomarkers for tobacco use since nicotine use can be from both tobacco and non-tobacco sources. This study aimed to provide an in-depth evaluation of the suitability of anabasine and anatabine as WBE biomarkers of tobacco and subsequently estimate their excretion factors for WBE applications. Pooled urine (n = 64) and wastewater samples (n = 277), collected between 2009 and 2019 in Queensland, Australia, were analyzed for nicotine and its metabolites (cotinine and hydroxycotinine), as well as anabasine and anatabine. Anabasine performed as the better biomarker, showing a similar per capita load in pooled urine (2.2 ± 0.3 µg/day/person) and wastewater samples (2.3 ± 0.3 µg/day/person), while the per capita load of anatabine in wastewater was 50% higher than its load in urine. It is estimated that 0.9 µg of anabasine was excreted per cigarette smoked. Triangulation of tobacco sales data and tobacco use estimated from either anabasine or cotinine showed that anabasine-based estimates were 5% higher than sales data, while cotinine-based estimates were between 2 and 28% higher. Our results provided concrete evidence to confirm the suitability of anabasine as a specific biomarker for monitoring tobacco use by WBE.

Novel proposed cutoff values for anatabine and anabasine in differentiating smokers from non-smokers.

OBJECTIVES: Anatabine and anabasine are two tobacco alkaloids used to differentiate between tobacco users and abstainers, including users of nicotine replacement therapy. Cutoff values (>2 ng/mL for both alkaloids) have not been revised since their implementation in 2002. These values may be too high, leading to increased likelihood of misclassification between smokers and abstainers. This results in major consequences, especially adverse outcomes of transplantation when smokers were incorrectly identified as being abstinent. This study proposes that a lower threshold for anatabine and anabasine will better distinguish tobacco users from non-users and thereby improve patients' care. DESIGN AND METHODS: A new and more sensitive analytical method by liquid chromatography-mass detection was developed to allow the quantification of low concentrations. Anatabine and anabasine were measured in urine samples of 116 self-reported daily smokers and 47 long-term non-smokers (confirmed by the analysis of nicotine and its metabolites). The best compromise between sensitivity and specificity allowed us to determine new cutoff values. RESULTS: The thresholds >0.097 ng/mL for anatabine and >0.236 ng/mL for anabasine were associated with a sensitivity of 97% (anatabine) and 89% (anabasine) and a specificity of 98% for both alkaloids. These cutoff values greatly increased the sensitivity given that it dropped to 75% (anatabine) and 47% (anabasine) when using the reference value (>2 ng/mL). CONCLUSIONS: The cutoff values >0.097 ng/mL for anatabine and >0.236 ng/mL for anabasine appear to better differentiate tobacco users from abstainers than the current reference threshold (>2 ng/mL for both alkaloids). It may considerably impact patients' care, especially in transplantation settings in which smoking abstinence is essential to avoid adverse outcomes of transplantation.

Anabasine and Anatabine Exposure Attributable to Cigarette Smoking: National Health and Nutrition Examination Survey (NHANES) 2013-2014.

Anabasine and anatabine are minor alkaloids in tobacco products and are precursors for tobacco-specific nitrosamines (TSNAs). The levels of these two compounds have been used to differentiate tobacco product sources, monitor compliance with smoking cessation programs, and for biomonitoring in TSNA-related studies. The concentrations of urinary anabasine and anatabine were measured in a representative sample of U.S. adults who smoked cigarettes (N = 770) during the 2013−2014 National Health and Nutrition Examination Survey (NHANES) study cycle, which was the first cycle where urinary anabasine and anatabine data became available. Weighted geometric means (GM) and geometric least squares means (LSM) with 95% confidence intervals were calculated for urinary anabasine and anatabine categorized by tobacco-use status [cigarettes per day (CPD) and smoking frequency] and demographic characteristics. Smoking ≥20 CPD was associated with 3.6× higher anabasine GM and 4.8× higher anatabine GM compared with smoking <10 CPD. Compared with non-daily smoking, daily smoking was associated with higher GMs for urinary anabasine (1.41 ng/mL vs. 6.28 ng/mL) and anatabine (1.62 ng/mL vs. 9.24 ng/mL). Urinary anabasine and anatabine concentrations exceeded the 2 ng/mL cut point in 86% and 91% of urine samples from people who smoke (PWS) daily, respectively; in comparison, 100% of them had serum cotinine concentrations greater than the established 10 ng/mL cut point. We compared these minor tobacco alkaloid levels to those of serum cotinine to assess their suitability as indicators of recent tobacco use at established cut points and found that their optimal cut point values would be lower than the established values. This is the first time that anabasine and anatabine are reported for urine collected from a U.S. population-representative sample of NHANES study participants, providing a snapshot of exposure levels for adults who smoked during 2013−2014. The results of this study serve as an initial reference point for future analysis of NHANES cycles, where changes in the national level of urinary anabasine and anatabine can be monitored among people who smoke to show the effect of changes in tobacco policy.

Anabasine-based measurement of cigarette consumption using wastewater analysis.

Community tobacco use can be monitored over time using wastewater-based epidemiological approaches by estimating the mass loads of nicotine and its metabolites, cotinine, or hydroxycotinine, in wastewater. However, due to the use of nicotine in smoking cessation products, other sources of nicotine contribute to cotinine and hydroxycotinine loads. The use of nicotine replacement therapies could vary in space and time and mask the true rates of tobacco consumption. Therefore, this work evaluated the content of tobacco specific markers, anatabine and anabasine, in cigarettes, in urine of smokers, and in wastewater. The results indicated that the anabasine content in both licit and illicit cigarettes in Australia is less variable than anatabine and is therefore considered a better measure of tobacco consumption. A study determining the excretion of tobacco-specific alkaloids of smoking and non-smoking volunteers gave an average urinary mass load of anabasine of 4.38 µg/L/person and a daily mass load of 1.13 µg/day/person. Finally, this was compared with the mass loads of anabasine from wastewater-based epidemiology data of 3 µg/day/person to estimate cigarette rates in a South Australian city: equivalent to 2.6 cigarettes/person/day. The rate of decline of cigarette use was greater when using anabasine as a measure of consumption compared with cotinine. This is the first study to estimate the rate of anabasine excretion, which can be used to estimate tobacco use independent of therapeutically prescribed nicotine.

Exposure characterization of three major insecticide lines in urine of young children in Japan-neonicotinoids, organophosphates, and pyrethroids.

The use of neonicotinoid (NEO) insecticides has increased over the past decade not only in Japan but also worldwide, while organophosphate (OP) and pyrethroid (PYR) insecticides are still conventionally used in agriculture and domestic pest control. However, limited data are currently available on the NEO exposure levels, especially in children, who are particularly vulnerable to environmental toxicants. Thus, the purpose of this study was to characterize the exposure to NEOs, as well as OPs and PYRs, in three-year-old Japanese children by assessing the range, distribution, and seasonal differences of the urinary concentrations of seven NEOs (acetamiprid, clothianidin, dinotefuran, thiacloprid, thiamethoxam, imidacloprid, and nitenpyram); four OP metabolites (dialkylphosphates [DAPs]), including dimethylphosphate, dimethylthiophosphate, diethylphosphate, and diethylthiophosphate; and three PYR metabolites (3-phenoxybenzoic acid, trans-chrysanthemumdicarboxylic acid, and 3-(2,2-dichlorovinyl)-2,2- dimethylcyclopropane carboxylic acid). Urine samples were collected from 223 children (108 males and 115 females) in the summer and winter months. The detection rates of NEOs were 58% for dinotefuran, 25% for thiamethoxam, 21% for nitenpyram, and <16% for all other NEOs. The median and maximum concentrations of the sum of the seven NEOs (ΣNEO) were 4.7 and 370.2nmol/g creatinine, respectively. Urinary ΣNEO, dimethylphosphate, and all PYR metabolite concentrations were significantly higher in the summer than in the winter (p<0.05). The creatinine-adjusted concentration of ΣNEO significantly correlated with those of all DAPs (p<0.05) but not with those of the PYR metabolites. Moreover, the NEO-detected group showed higher urinary ΣDAP (sum of four OP metabolites) concentrations than the group without NEO detection. These findings suggest that children in Japan are environmentally exposed to the three major insecticide lines, and that the daily exposure sources of NEOs are common to those of OPs.

Quantitation of the Minor Tobacco Alkaloids Nornicotine, Anatabine, and Anabasine in Smokers' Urine by High Throughput Liquid Chromatography-Mass Spectrometry.

Nicotine is the most abundant alkaloid in tobacco accounting for 95% of the alkaloid content. There are also several minor tobacco alkaloids; among these are nornicotine, anatabine, and anabasine. We developed and applied a 96 well plate-based capillary LC-tandem mass spectrometry method for the analysis of nornicotine, anatabine, and anabasine in urine. The method was validated with regard to accuracy and precision. Anabasine was quantifiable to low levels with a limit of quantitation (LOQ) of 0.2 ng/mL even when nicotine, which is isobaric, is present at concentrations >2500-fold higher than anabasine. This attribute of the method is important since anatabine and anabasine in urine have been proposed as biomarkers of tobacco use for individuals using nicotine replacement therapies. In the present study, we analyzed the three minor tobacco alkaloids in urine from 827 smokers with a wide range of tobacco exposures. Nornicotine (LOQ 0.6 ng/mL) was detected in all samples, and anatabine (LOQ, 0.15 ng/mL) and anabasine were detected in 97.7% of the samples. The median urinary concentrations of nornicotine, anatabine, and anabasine were 98.9, 4.02, and 5.53 ng/mL. Total nicotine equivalents (TNE) were well correlated with anatabine (r(2) = 0.714) and anabasine (r(2) = 0.760). TNE was most highly correlated with nornicotine, which is also a metabolite of nicotine. Urine samples from a subset of subjects (n = 110) were analyzed for the presence of glucuronide conjugates by quantifying any increase in anatabine and anabasine concentrations after ß-glucuronidase treatment. The median ratio of the glucuronidated to free anatabine was 0.74 (range, 0.1 to 10.9), and the median ratio of glucuronidated to free anabasine was 0.3 (range, 0.1 to 2.9). To our knowledge, this is the largest population of smokers for whom the urinary concentrations of these three tobacco alkaloids has been reported.

Neonicotinoid concentrations in urine from chronic kidney disease patients in the North Central Region of Sri Lanka.

OBJECTIVES: Neonicotinoid insecticides have been widely used around the world since the 1990s. Reports have been made since the 1990s of rice paddy farmers in the North Central Region (NCR) of Sri Lanka suffering from chronic kidney disease with unknown etiology (CKDu). A preliminary evaluation of the exposure of local farmers in the NCR of Sri Lanka to neonicotinoids was performed. METHODS: We analyzed neonicotinoid and neonicotinoid metabolite concentrations in spot urine samples. We selected 40 samples, 10 from farmers with CKDu and 10 from controls from each of two areas, Medawachchiya and Girandurukotte. RESULTS: Imidacloprid and desmethyl-acetamiprid were found at significantly higher concentrations in the control samples (with medians of 51 ng/l and 340 ng/l, respectively) than in the CKDu samples (medians of 15 ng/l and 150 ng/l, respectively) when the results were not adjusted for the creatinine contents. None of the six compounds that were measured in the urine samples were found at significantly higher concentrations in the CKDu samples than in the control samples. None of the neonicotinoid concentrations in the samples analyzed in this study exceeded the concentrations that have been found in samples from the general population of Japan. CONCLUSIONS: Farmers (both with and without CKDu) living in CKDu-endemic areas in the NCR of Sri Lanka are exposed to lower neonicotinoid concentrations than non-occupationally exposed residents of Japan.

A high-throughput robotic sample preparation system and HPLC-MS/MS for measuring urinary anatabine, anabasine, nicotine and major nicotine metabolites.

BACKGROUND: Most sample preparation methods characteristically involve intensive and repetitive labor, which is inefficient when preparing large numbers of samples from population-scale studies. METHODS: This study presents a robotic system designed to meet the sampling requirements for large population-scale studies. Using this robotic system, we developed and validated a method to simultaneously measure urinary anatabine, anabasine, nicotine and seven major nicotine metabolites: 4-Hydroxy-4-(3-pyridyl)butanoic acid, cotinine-N-oxide, nicotine-N-oxide, trans-3'-hydroxycotinine, norcotinine, cotinine and nornicotine. We analyzed robotically prepared samples using high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry in positive electrospray ionization mode using scheduled multiple reaction monitoring (sMRM) with a total runtime of 8.5 min. RESULTS: The optimized procedure was able to deliver linear analyte responses over a broad range of concentrations. Responses of urine-based calibrators delivered coefficients of determination (R(2)) of >0.995. Sample preparation recovery was generally higher than 80%. The robotic system was able to prepare four 96-well plate (384 urine samples) per day, and the overall method afforded an accuracy range of 92-115%, and an imprecision of <15.0% on average. CONCLUSIONS: The validation results demonstrate that the method is accurate, precise, sensitive, robust, and most significantly labor-saving for sample preparation, making it efficient and practical for routine measurements in large population-scale studies such as the National Health and Nutrition Examination Survey (NHANES) and the Population Assessment of Tobacco and Health (PATH) study.

Reference interval determination for anabasine: a biomarker of active tobacco use.

Laboratory detection of nicotine exposure is important for establishing eligibility for organ transplant and elective surgery. Nicotine testing is also used to verify compliance with nicotine replacement therapies (NRT), smoking cessation programs and for life insurance purposes. Nicotine metabolites, such as cotinine and trans-3'-hydroxycotinine, are used as biomarkers of nicotine exposure. For some clinical applications, it is important to distinguish between active use of tobacco products versus NRT. Anabasine is a tobacco alkaloid that has been used as a biomarker of active tobacco use. However, the use of anabasine as an insecticide, and its presence in consumables other than nicotine products, suggests that anabasine may not be specific to tobacco use/exposure. Here, we determine the reference interval for anabasine in the urine of nonsmokers and compare it to the range of anabasine concentrations observed in the presence or absence of nicotine metabolites.

Qualitative profiling and quantification of neonicotinoid metabolites in human urine by liquid chromatography coupled with mass spectrometry.

Neonicotinoid pesticides have been widely applied for the production of fruits and vegetables, and occasionally detected in conventionally grown produce. Thus oral exposure to neonicotinoid pesticides may exist in the general population; however, neonicotinoid metabolites in human body fluids have not been investigated comprehensively. The purpose of this study is the qualitative profiling and quantitative analysis of neonicotinoid metabolites in the human spot urine by liquid chromatography coupled with mass spectrometry (LC/MS). Human urine samples were collected from three patients suspected of subacute exposure to neonicotinoid pesticides. A qualitative profiling of urinary metabolites was performed using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) with a database of nominal molecular weights of 57 known metabolites of three neonicotinoid pesticides (acetamiprid, Imidacloprid, and clothianidin), as well as the parent compounds. Then a quantitative analysis of selected urinary metabolites was performed using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with a standard pesticide and metabolite, which were detected by the qualitative profiling. The result of qualitative profiling showed that seven metabolites, i.e. an acetamiprid metabolite, N-desmethyl-acetamiprid; three Imidacloprid metabolites, 5-hydroxy-Imidacloprid, 4,5-dihydroxy-imidacloprid, 4,5-dehydro-Imidacloprid; a common metabolite of acetamiprid and Imidacloprid, N-(6-chloronicotinoyl)-glycine; and two clothianidin metabolites, N-desmethyl-clothianidin, N-(2-(methylsulfanyl)thiazole-5-carboxyl)-glycine, as well as acetamiprid, were detected in the urine of three cases. The result of the quantitative analysis showed N-desmethyl-acetamiprid was determined in the urine of one case, which had been collected on the first visit, at a concentration of 3.2 ng/mL. This is the first report on the qualitative and quantitative detection of N-desmethyl-acetamiprid in the human urine. The results suggest that the one case with detection of N-desmethyl-acetamiprid was exposed to acetamiprid through the consumption of contaminated foods. Urinary N-desmethyl-acetamiprid, as well as 5-hydroxy-Imidacloprid and N-desmethyl-clothianidin, may be a good biomarker for neonicotinoid exposure in humans and warrants further investigation.

A one-year monitoring of nicotine use in sport: frontier between potential performance enhancement and addiction issues.

Tobacco consumption is a global epidemic responsible for a vast burden of disease. With pharmacological properties sought-after by consumers and responsible for addiction issues, nicotine is the main reason of this phenomenon. Accordingly, smokeless tobacco products are of growing popularity in sport owing to potential performance enhancing properties and absence of adverse effects on the respiratory system. Nevertheless, nicotine does not appear on the 2011 World Anti-Doping Agency (WADA) Prohibited List or Monitoring Program by lack of a comprehensive large-scale prevalence survey. Thus, this work describes a one-year monitoring study on urine specimens from professional athletes of different disciplines covering 2010 and 2011. A method for the detection and quantification of nicotine, its major metabolites (cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide) and minor tobacco alkaloids (anabasine, anatabine and nornicotine) was developed, relying on ultra-high pressure liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-TQ-MS/MS). A simple and fast dilute-and-shoot sample treatment was performed, followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. After method validation, assessing the prevalence of nicotine consumption in sport involved analysis of 2185 urine samples, accounting for 43 different sports. Concentrations distribution of major nicotine metabolites, minor nicotine metabolites and tobacco alkaloids ranged from 10 (LLOQ) to 32,223, 6670 and 538 ng/mL, respectively. Compounds of interest were detected in trace levels in 23.0% of urine specimens, with concentration levels corresponding to an exposure within the last three days for 18.3% of samples. Likewise, hypothesizing conservative concentration limits for active nicotine consumption prior and/or during sport practice (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N'-oxide, cotinine-N-oxide, anabasine, anatabine and nornicotine) revealed a prevalence of 15.3% amongst athletes. While this number may appear lower than the worldwide smoking prevalence of around 25%, focusing the study on selected sports highlighted more alarming findings. Indeed, active nicotine consumption in ice hockey, skiing, biathlon, bobsleigh, skating, football, basketball, volleyball, rugby, American football, wrestling and gymnastics was found to range between 19.0 and 55.6%. Therefore, considering the adverse effects of smoking on the respiratory tract and numerous health threats detrimental to sport practice at top level, likelihood of smokeless tobacco consumption for performance enhancement is greatly supported.

Nicotiana glauca (tree tobacco) intoxication--two cases in one family.

We present two cases of rare human poisoning in one family following ingestion of cooked leaves from the tobacco tree plant, Nicotiana glauca. The toxic principle of N. glauca, anabasine (C10H14N2), is a small pyridine alkaloid, similar in both structure and effects to nicotine, but appears to be more potent in humans. A 73-year-old female tourist from France, without remarkable medical history, collapsed at home following a few hours long prodrome of dizziness, nausea, vomiting, and malaise. The symptoms developed shortly after eating N. glauca cooked leaves that were collected around her daughter's house in Jerusalem and mistaken for wild spinach. She was found unconscious, with dilated pupils and extreme bradycardia. Following resuscitation and respiratory support, circulation was restored. However, she did not regain consciousness and died 20 days after admission because of multi-organ failure. Anabasine was identified by gas chromatography/mass spectrometry method in N. glauca leaves and in the patient's urine. Simultaneously, her 18-year-old grandson developed weakness and myalgia after ingesting a smaller amount of the same meal. He presented to the same emergency room in a stable condition. His exam was remarkable only for sinus bradycardia. He was discharged without any specific treatment. He recovered in 24 h without any residual sequelae. These cases raise an awareness of the potential toxicity caused by ingestion of tobacco tree leaves and highlight the dangers of ingesting botanicals by lay public. Moreover, they add to the clinical spectrum of N. glauca intoxication.

Determination of nicotine, anabasine, and cotinine in urine and saliva samples using single-drop microextraction.

A simple, sensitive, and inexpensive singe-drop microextraction (SDME) followed by gas chromatography and flame-ionization detection (GC-FID) was developed for determination of nicotine, anabasine, and cotinine in human urine and saliva samples. The target compounds were extracted from alkaline aqueous sample solution into an organic acceptor drop suspended on the tip of a 25-µL GC microsyringe in the aqueous sample solution. This microsyringe was also used for direct injection after extraction. Under optimized experimental conditions, calibration plots were found to be linear in the range of 0.5-25.0, 0.5-65.0, and 0.5-45.0mgL(-1) for nicotine, anabasines and cotinine, respectively. The method detection limit values were in the range of 0.33-0.45mgL(-1). Intra-day and inter-day precisions for peak area ratios were in the range of 1.3-9.2% and 2.0-7.0%, respectively. The proposed procedure was successfully applied to the determination of analytes in spiked urine and saliva samples with satisfactory results. The mean relative recoveries of spiked water samples ranged over 71.2-111.0%, with relative standard deviations varying from 2.3% to 10.0%.

Quantitation of nicotine, its metabolites, and other related alkaloids in urine, serum, and plasma using LC-MS-MS.

We describe a method for the quantitative analysis of nicotine, cotinine, trans-3'-hydroxy cotinine, nornicotine, and anabasine in urine, serum, and plasma using liquid chromatography-tandem mass spectrometry. A mix of deuterium-labeled internal standards (IS) is added to a specimen aliquot. The aliquot is extracted using mixed-mode solid phase extraction and eluted into an autosampler vial for injection into an LC-MS-MS system. An Atlantis silica column is used for LC separation in hydrophilic interaction mode. Tandem mass spectrometry detection is performed in positive ion mode with electrospray ionization and two multiple reaction monitoring (MRM) transitions monitored for each analyte and IS.

Determination of nicotine, cotinine, and related alkaloids in human urine and saliva by automated in-tube solid-phase microextraction coupled with liquid chromatography-mass spectrometry.

A simple, rapid and sensitive method for the determination of nicotine, cotinine, nornicotine, anabasine, and anatabine in human urine and saliva was developed. These compounds were analyzed by on-line in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-mass spectrometry (LC-MS). Nicotine, cotinine and related alkaloids were separated within 7 min by high performance liquid chromatography (HPLC) using a Synergi 4u POLAR-RP 80A column and 5 mM ammonium formate/methanol (55/45, v/v) as a mobile phase at a flow-rate of 0.8 mL/min. Electrospray ionization conditions in the positive ion mode were optimized for MS detection of these compounds. The optimum in-tube SPME conditions were 25 draw/eject cycles with a sample size of 40 microL using a CP-Pora PLOT amine capillary column as the extraction device. The extracted compounds could be desorbed easily from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC-MS method, the calibration curves were linear in the concentration range of 0.5-20 ng/mL of nicotine, cotinine and related compounds in urine and saliva, and the detection limits (S/N=3) were 15-40 pg/mL. The method described here showed 20-46-fold higher sensitivity than the direct injection method (5 microL injection). The within-run and between-day precision (relative standard deviations) were below 4.7% and 11.3% (n=5), respectively. This method was applied successfully to analysis of urine and saliva samples without interference peaks. The recoveries of nicotine, cotinine and related compounds spiked into urine and saliva samples were above 83%, and the relative standard deviations were below 7.1%. This method was used to analyze urinary and salivary levels of these compounds in nicotine intake and smoking.

Specific detection of anabasine, nicotine, and nicotine metabolites in urine by liquid chromatography-tandem mass spectrometry.

The sensitive and specific detection of nicotine, its metabolites, and the tobacco alkaloid, anabasine, is useful in evaluating the success of smoking cessation treatments and detecting tobacco use, passive exposure, and nontobacco nicotine exposure in potential transplant recipients, insurance clients, and elective surgical patients. Rapid sample preparation and extended high-performance liquid chromatographic separation of tobacco alkaloids and metabolites was interfaced with tandem mass spectrometry. By using deuterated internal standards and appropriate confirmatory ion mass transitions, direct injection of centrifugally clarified urine was possible. The method had excellent precision, limit of quantitation, and linearity. The rigorous separation method revealed an interferent of nicotine that had coeluted with anabasine in more rapid chromatography and that may result in tobacco use misclassification. The method provides more specific detection of tobacco exposure and illustrates the potential of centrifugal clarification for sample preparation in the detection of multiple analytes in urine.

Simultaneous and sensitive measurement of anabasine, nicotine, and nicotine metabolites in human urine by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Determination of nicotine metabolism/pharmacokinetics provides a useful tool for estimating uptake of nicotine and tobacco-related toxicants, for understanding the pharmacologic effects of nicotine and nicotine addiction, and for optimizing nicotine dependency treatment. METHODS: We developed a sensitive method for analysis of nicotine and five major nicotine metabolites, including cotinine, trans-3'-hydroxycotinine, nicotine-N'-oxide, cotinine-N-oxide, and nornicotine, in human urine by liquid chromatography coupled with a TSQ Quantum triple quadrupole tandem mass spectrometer (LC/MS/MS). Urine samples to which deuterium-labeled internal standards had been added were extracted with a simple solid-phase extraction procedure. Anabasine, a minor tobacco alkaloid, was also included. RESULTS: The quantification limits of the method were 0.1-0.2 microg/L, except for nicotine (1 microg/L). Cotinine-N-oxide, trans-3'-hydroxycotinine, nicotine, and anabasine in urine were almost completely recovered by the solid-phase extraction, whereas the mean extraction recoveries of nicotine-N'-oxide, cotinine, and nornicotine were 51.4%, 78.6%, and 78.8%, respectively. This procedure provided a linearity of three to four orders of magnitude for the target analytes: 0.2-400 microg/L for nicotine-N'-oxide, cotinine-N-oxide, and anabasine; 0.2-4000 microg/L for cotinine, nornicotine, and trans-3'-hydroxycotinine; and 1.0-4000 microg/L for nicotine. The overall interday method imprecision and recovery were 2.5-18% and 92-109%, respectively. CONCLUSIONS: This sensitive LC/MS/MS procedure can be used to determine nicotine metabolism profiles of smokers, people during nicotine replacement therapy, and passively exposed nonsmokers. This method avoids the need for a time-consuming and labor-intensive sample enrichment step and thus allows for high-throughput sample preparation and automation.

Anabasine and anatabine as biomarkers for tobacco use during nicotine replacement therapy.

In this study we determined urine concentration of the tobacco alkaloids anabasine and anatabine, nicotine and its metabolites cotinine, and nornicotine in 99 cigarette smokers and 205 smokeless tobacco users. We also investigated the possibility that anabasine and anatabine can be used as biomarkers for tobacco use during nicotine replacement therapy. Urine samples and data on self-reported tobacco use were obtained from subjects enrolled in tobacco cessation programs. Urine concentrations of tobacco alkaloids and metabolites were measured and correlated with self-reported tobacco use. Concentrations of anabasine and anatabine were used to validate abstinence in smokeless tobacco users who used nicotine gum as part of the therapy. Correlations of alkaloid concentration with self-reported tobacco use before treatment ranged from fair to poor. In subjects abstaining from smokeless tobacco but using nicotine gum, anabasine and anatabine levels were below the cut-point of 2 ng/ml despite high concentrations of nicotine and cotinine resulting from nicotine gum use. Anabasine and anatabine concentrations in urine can be used to validate abstinence or measure the extent of tobacco use in persons undergoing nicotine replacement therapy.