Article pubs.acs.org/crt
Quantitation of the Minor Tobacco Alkaloids Nornicotine, Anatabine, and Anabasine in Smokers’ Urine by High Throughput Liquid Chromatography−Mass Spectrometry Linda B. von Weymarn,† Nicole M. Thomson,† Eric C. Donny,§ Dorothy K. Hatsukami,‡ and Sharon E. Murphy*,† †
Department of Biochemistry Molecular Biology and Biophysics and Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, United States ‡ Masonic Cancer Center and Department of Psychiatry, University of Minnesota, Minneapolis, Minnesota 55455, United States § Department of Psychology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States S Supporting Information *
ABSTRACT: 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 (r2 = 0.714) and anabasine (r2 = 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 β-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.
■
INTRODUCTION Tobacco use is the number one preventable cause of death in the United States and is responsible for one in five deaths.1 The complete cessation of tobacco use would be the ideal solution to this public health challenge. However, due to the addictive properties of the nicotine present in tobacco, maintaining abstinence from tobacco-use is challenging. Forty-three percent of daily smokers make a quit attempt every year, yet few are successful.2 In the United States, 17.8% of adults still smoke, and 4% use smokeless tobacco products.2,3 It has been suggested that if the nicotine content of cigarettes were significantly reduced, then this lethal product would become nonaddictive.4 In 2009, the Food and Drug Administration (FDA) was granted the authority to reduce but not eliminate nicotine in tobacco. In collaboration with others, we recently published the © XXXX American Chemical Society
results of a 6-week randomized clinical trial in which nicotine exposure from reduced-nicotine cigarettes was compared to exposure from standard nicotine content cigarettes.5 Exposure, quantified by the urinary concentration of total nicotine equivalents (TNE, the sum of nicotine and six metabolites) was significantly reduced in users of cigarettes with nicotine content ≤2.4 mg/g compared to those using cigarettes with 15.8 mg of nicotine per g, a level comparable to that in a Marlboro cigarette.6 Nicotine dependence was also reduced. These data suggest that if these effects are replicated in longer term studies, potentially with greater compliance to the use of low nicotine content cigarettes, then reduction of nicotine in cigarettes could lead to a reduction in smoking prevalence. The Received: December 21, 2015
A
DOI: 10.1021/acs.chemrestox.5b00521 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
Article
Chemical Research in Toxicology
and 0.33 pg/μL. Linear calibration curves (3 independent sets) were prepared keeping the concentration of the d4-compounds constant (25 pg/μL) and varying the concentration of nornicotine, anatabine, and anabasine (25 pg/μL, 2.5 pg/μL, 0.25 pg/μL, 0.025 pg/μL, and 0 pg/ μL). The limit of detection and relative response of the instrument were determined from these calibration curves. The calibration curves were run prior to each series of LC/MS/MS analyses. The limit of quantitation and linearity of the assay were determined by analyzing a series of samples with a 400-fold range of anatabine, anabasine, and nornicotine concentrations. These samples were prepared either by adding standard minor alkaloids solutions to a nonsmoker’s urine or by serial dilution of a positive control smoker’s urine sample with a nonsmoker’s urine. The concentrations of anatabine, anabasine, and nornicotine in the positive control sample were previously determined by a well-established GC-MS method.8,19 Urine Samples. The urine samples used in this study are the baseline samples from smokers who participated in a randomized trial of reduced nicotine content cigarettes. We previously analyzed the TNE concentration of these samples.5 At baseline, the participants (n = 839, 38% Black, 51% White, and 5% Hispanic) were smoking their usual brand of cigarette and did not report using any other nicotine containing products in more than 9 of the last 30 days. Eligibility criteria for the trial included an age of 18 years, use of at least 5 cigarettes per day, either expired carbon monoxide levels >8 ppm or urinary cotinine concentration greater than 100 ng/mL at the screening visit. Among the 839 baseline urine samples, 11 with baseline cotinine levels less than 100 ng/mL, as they did not meet one of the screening criteria of the clinical trial (TNE of these samples was 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 the confirmation of abstinence in individuals using nicotine replacement therapies.9 More recently, we have proposed that these minor alkaloids also may be used to E
DOI: 10.1021/acs.chemrestox.5b00521 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
Article
Chemical Research in Toxicology
As previously reported, the relative amounts of nornicotine, anabasine, and anatabine in the urine of smokers were quite different from their relative levels in tobacco. In the first analysis of these alkaloids in smokers’ urine (n = 22), the ratio of the mean concentrations of nornicotine to anabasine to anatabine was 6.5 to 1 to 1.5.25 However, the most abundant of these minor tobacco alkaloids in the 50 top selling cigarettes in the United States is anatabine, and the mean ratio of nornicotine to anabasine to anatabine is 5.8 to 1.0 to 7.5.11 In the smokers of our study, the ratio of the median levels of the three alkaloids was 10.6 to 1 to 0.8. The difference in the distribution of these minor alkaloids in tobacco and urine is in part due to differences in the transfer of the alkaloid from tobacco to the smoke.26 There is little data on the smoke level of these tobacco alkaloids in commercial cigarettes. The amounts of nornicotine, anabasine, and anatabine in smoke from reference cigarettes are 73, 7, and 18 μg/g tobacco, respectively.27 The relative levels of nornicotine and anabasine in tobacco smoke are similar to their relative concentrations in urine. The lower concentration of anatabine than anabasine in most smokers’ urine may be due to the greater metabolism of anatabine. We confirm here that one significant pathway of anatabine metabolism is glucuronidation, but other pathways are also likely to contribute. Both anatabine and anabasine were detected in 97.7% of the smokers’ urine samples in our study, and only 6 samples had nondetectable levels of either of these tobacco alkaloids. Anatabine and anabasine were both well correlated with TNE. However, for a specific concentration of the minor alkaloid a range of TNE was observed. For example, whereas no smoker with TNE below 10 had either anatabine or anabasine levels >2 ng/mL, TNE concentrations ranged from 2.5 to 69.1 nmol/mL in smokers with both anatabine and anabasine concentrations below 2 ng/mL. The association of a wide range of TNE levels among smokers with similar urinary anabasine and anatabine concentrations may be due to differences in the amount of the minor alkaloids relative to nicotine present in the cigarettes smoked. Alternatively, the relatively high TNE in some smokers with low levels of anatabine or anabasine may be due to the variability in the time of the last cigarette and individual differences between the half-life of nicotine and its metabolites compared to those of the minor alkaloids. Nicotine metabolism and clearance are well studied.20,24,28 However, there is little to no data on the metabolism of anatabine and anabasine in smokers. A study a number of years ago suggests that there is little metabolism of anabasine; when anabasine was administered orally, about 70% of the dose was excreted unchanged in the urine.29
Figure 4. Total nicotine equivalents of smokers with urinary anatabine and anabasine concentrations
