Combined analysis of N′-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in the urine of cigarette smokers and e-cigarette users

Highlights

• Urinary total NNN and total NNAL are important human carcinogen biomarkers.

• An LC–ESI⁺–MS/MS method was developed for their combined analysis in human urine.

• Use of monitor amine [pyridine-D₄]nornicotine assessed artifact formation.

• Smokers had significantly higher levels of total NNN and NNAL than e-cigarette users.

Abstract

A liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI⁺–MS/MS) method for the analysis of the tobacco-specific carcinogens N′-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and their glucuronides (total NNN and total NNAL) in human urine was developed. The method has excellent accuracy and intra-day and inter-day precision, and limits of quantitation of 0.015 and 0.075 pmol/mL urine, respectively, for total NNN and total NNAL. A unique aspect of this method is internal assessment of possible artifactual formation of NNN by inclusion of the monitor amine [pyridine-D₄]nornicotine. We found that artifactual formation of NNN comprised only 2.5% of the measured amounts of total NNN in urine of cigarette smokers, under our conditions using ammonium sulfamate as an inhibitor of nitrosation. The method was applied to urine samples from cigarette smokers and e-cigarette users. Levels of total NNN and total NNAL in the urine of cigarette smokers averaged 0.060 ± 0.035 pmol/mL and 2.41 ± 1.41 pmol/mL urine, (N = 38), respectively, which were both significantly greater than in the urine of 27 e-cigarette users.

Introduction

The tobacco-specific nitrosamines N′-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (Fig.1) are present in all tobacco products and are considered “carcinogenic to humans” by the International Agency for Research on Cancer [1], [2]. NNN and NNK are formed during the processing and curing of tobacco. During cigarette smoking, NNN and NNK are transferred through the smoke to both the oral tissues and lungs of smokers [3]. Each cigarette typically delivers about 100–150 ng NNN and 50–100 ng NNK to the smoker [4]. NNN causes oral cavity and esophageal cancer in rats, and tumors of the respiratory tract in mice, hamsters, and mink. NNK is a powerful organoselective lung carcinogen in rats, mice, and hamsters while also inducing tumors at other sites including the pancreas and nasal mucosa [5], [6], [7]. Because of their potent carcinogenicity and tobacco- specificity, NNN and NNK are widely acknowledged as important causes of cancer in tobacco users.

Human exposure to NNN and NNK can be assessed by analysis of urine. Unchanged NNN as well as its pyridine-N-glucuronide are excreted in the urine [8], [9], [10], [11], [12]. Unchanged NNK is not generally detected in human urine. Rather, its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, Fig. 1) and its O- and N-glucuronides are present in the urine of all smokers [3], [13], [14]. Total NNN and total NNAL (the sum of the free compounds and their glucuronide metabolites) are useful biomarkers of NNN and NNK exposure. Total NNAL in particular has been quantified in thousands of urine samples from smokers [3]. Total NNN and total NNAL are also risk biomarkers; levels of urinary total NNN have been strongly related to the risk of esophageal cancer and total NNAL to the risk of lung cancer in nested case-control studies carried out within a prospective epidemiology study in Shanghai [15], [16], [17] .

Accurate assessment of total NNN and total NNAL is critical in cancer prevention strategies related to tobacco products. A reliable combined assay for quantifying these metabolites in human urine would therefore be an important addition to a panel of carcinogen exposure and cancer risk biomarkers. Our group was the first to describe an assay for free NNN and its glucuronide, as well as free and glucuronidated N′-nitrosoanabasine (NAB) and N′-nitrosoanatabine (NAT) in human urine [8]. The levels of total NNN were compared to those of total NNAL, determined separately. Two research groups have subsequently described combined assays for total NNAL, total NNN, total NAB, and total NAT in human urine [10], [11], [18]. A method for total NNN has also been briefly summarized [19].

The two studies by Kavvadias et al. mention the problem of potential artifactual formation of NNN, but this was not addressed in the study by Xia et al. We are aware from years of experience in the trace analysis of NNN that artifact formation can present problems because all tobacco products contain nornicotine, which is readily transferred to the saliva and urine of smokers and can be easily nitrosated to form NNN [20]. We have addressed and excluded this problem in our previous studies [8], [12], [16], [21], [22], [23], [24]. However, one must be continually aware of the potential for artifactual formation of NNN, particularly when low levels such as those expected in the urine of e-cigarette users are being analyzed. Therefore, in the study described here for analysis of total NNN and total NNAL in urine, we carefully monitored for artifactual formation of NNN by addition of [pyridine-D₄] nornicotine to each urine sample. We have applied our method to the analysis of total NNN and total NNAL in the urine of cigarette smokers and users of e-cigarettes. The use of e-cigarettes has increased dramatically while they remain completely unregulated and little information is available regarding their toxicological effects [25], [26], [27]. It is possible that NNN could be formed endogenously in e-cigarette users by the reaction of nornicotine, a metabolite and common contaminant of nicotine, with salivary nitrite.