von Weymarn Linda B, Thomson Nicole M, Donny Eric C, Hatsukami Dorothy K, Murphy Sharon E
Department of Biochemistry Molecular Biology and Biophysics and Masonic Cancer Center, University of Minnesota , Minneapolis, Minnesota 55455, United States.
Department of Psychology, University of Pittsburgh , Pittsburgh, Pennsylvania 15260, United States.
Chem Res Toxicol. 2016 Mar 21;29(3):390-7. doi: 10.1021/acs.chemrestox.5b00521. Epub 2016 Feb 12.
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.
尼古丁是烟草中含量最丰富的生物碱，占生物碱总量的95%。烟草中还含有几种次要的生物碱，其中包括去甲烟碱、降烟碱和新烟草碱。我们开发并应用了一种基于96孔板的毛细管液相色谱 - 串联质谱法来分析尿液中的去甲烟碱、降烟碱和新烟草碱。该方法在准确性和精密度方面得到了验证。即使存在与新烟草碱等压的尼古丁，且其浓度比新烟草碱高2500倍以上，新烟草碱仍可低水平定量，定量限（LOQ）为0.2 ng/mL。该方法的这一特性很重要，因为尿液中的降烟碱和新烟草碱已被提议作为使用尼古丁替代疗法的个体烟草使用的生物标志物。在本研究中，我们分析了827名烟草暴露程度广泛的吸烟者尿液中的三种次要烟草生物碱。所有样本中均检测到了去甲烟碱（LOQ 0.6 ng/mL），97.7%的样本中检测到了降烟碱（LOQ，0.15 ng/mL）和新烟草碱。去甲烟碱、降烟碱和新烟草碱的尿中浓度中位数分别为98.9、4.02和5.53 ng/mL。总尼古丁当量（TNE）与降烟碱（r(2) = 0.714）和新烟草碱（r(2) = 0.760）高度相关。TNE与去甲烟碱相关性最高，而去甲烟碱也是尼古丁的一种代谢物。通过定量β - 葡萄糖醛酸酶处理后降烟碱和新烟草碱浓度的任何增加，分析了一部分受试者（n = 110）的尿液样本中葡萄糖醛酸结合物的存在情况。葡萄糖醛酸化降烟碱与游离降烟碱的中位数比值为0.74（范围为0.1至10.9），葡萄糖醛酸化新烟草碱与游离新烟草碱的中位数比值为0.3（范围为0.1至2.9）。据我们所知，这是已报道这三种烟草生物碱尿中浓度的最大吸烟者群体。
