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

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 (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 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.

Figure 1

LC-ESI-MS/MS chromatograms from analysis of a smoker's urine sample for anatabine (2.8 ng/ml), nornicotine (31.6 ng/ml) and anabasine (3.3 ng/ml), with select reaction monitoring for m/z 161 → 144, m/z 149→130 and m/z 163 → 118, respectively. The 100% abundance for m/z 161 → 144, is 5 × 10⁴ compared to 7.8 × 10³ for the other mass transitions. The total nicotine equivalents concentration of this urine sample was 16.4 nmol/ml. The nicotine concentration was 606 ng/ml.

Figure 2

LC-ESI-MS/MS analysis of anabasine by selected reaction monitoring of m/z 163→118 (upper panels) and m/z 163→144 (lower panels). A) A smoker's sample containing 0.38 ng/ml anabasine and 38 ng/ml nicotine was analyzed twice, before and after the addition of 0.50 ng/ml anabasine to the urine, the chromatograms obtained for each analyses are overlaid. [The ratios of the peak area of the anabasine to the d₄-anabasine internal standard were 0.042 and 0.098 in the two samples] B) The sample with the added anabasine was then co-injected with nicotine at a concentration 2000 times that of anabasine.

Figure 3

Distribution of urinary anatabine and anabasine concentrations in smokers. Samples with anatabine and anabasine concentrations from the limit of quantitation (LOQ) to 20 ng/ml are included in the main figure. The insert shows the distribution for all 827 subjects. LOQ: Anabasine, 0.2ng/ml, Anatabine, 0.15 ng/ml.

Figure 4

Total nicotine equivalents of smokers with urinary anatabine and anabasine concentrations <2 ng/ml, or < 1 ng/ml anatabine, or anabasine < 1 ng/ml. The dashed lines indicate the median total nicotine concentrations in the 3 groups (19.1, 17.1 and 12.7nmol/ml).