Elsevier

Journal of Chromatography A

Volume 1216, Issue 45, 6 November 2009, Pages 7899-7905
Journal of Chromatography A

Rapid and sensitive gas chromatography–ion-trap tandem mass spectrometry method for the determination of tobacco-specific N-nitrosamines in secondhand smoke

https://doi.org/10.1016/j.chroma.2009.09.020Get rights and content

Abstract

Tobacco-specific nitrosamines (TSNAs) are some of the most potent carcinogens in tobacco and cigarette smoke. Accurate quantification of these chemicals is needed to help assess public health risks. We developed and validated a specific and sensitive method to measure four TSNAs adsorbed to model surfaces and secondhand smoke (SHS) particles using gas chromatography–ion-trap tandem mass spectrometry. In an 18-m3 room-sized chamber, a smoking machine generated realistic concentrations of SHS that were actively sampled on Teflon-coated fiber glass (TCFG) filters, and passively sampled on cellulose substrates. A simple solid–liquid extraction protocol using methanol as solvent was successfully applied to both substrates with recoveries ranging from 85 to 115%. For each TSNA, tandem MS parameters were optimized and the major fragmentation pathways were elucidated. The method showed excellent performance, with a linear dynamic range from 2 to 1000 ng mL−1, low detection limits (S/N > 3) of 30–300 pg mL−1 and precision with experimental errors below 10% for all compounds. Moreover, no interfering peaks were observed, indicating a high selectivity of MS/MS without the need for a sample clean-up step. This method provides a suitable analytical tool to detect and quantify traces of TSNA in indoor environments polluted with SHS.

Introduction

Secondhand tobacco smoke (SHS), also called environmental tobacco smoke (ETS) or passive smoke, consists primarily of diluted and dispersed sidestream tobacco smoke that is emitted by smoldering cigarettes. In the US every year, exposure to SHS kills more than 3000 adult non-smokers from lung cancer, approximately 46,000 from coronary heart disease and an estimated 430 newborns from sudden infant death syndrome [1]. SHS contains many mutagenic and carcinogenic chemicals [2]. Of these, tobacco-specific nitrosamines (TSNAs) are among the most potent carcinogens identified in tobacco and its smoke [3], [4]. They are formed by nitrosation of nicotine and related tobacco alkaloids during aging, curing, fermentation and combustion [5], [6], [7]. N-Nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (NNAL) are the most important of the TSNAs because of their abundance and strong carcinogenicity [4], [8], [9]. NNK and NNN are classified as human carcinogens [10].

To assess exposure of non-smokers to TSNAs in the indoor environment, it is necessary to develop a fast, sensitive, and selective analytical method that can accurately determine low levels of these compounds in SHS. The most widely used method for TSNA analysis has been gas chromatography (GC) coupled with a thermal energy analyzer (TEA) [11], [12], [13]. However, the GC–TEA method has two main disadvantages: (1) it cannot differentiate the co-eluted nitroso-compounds although it is nitroso-specific, and (2) it requires extensive sample preparation, including pre-concentration and clean-up using liquid–liquid (L–L) extraction and/or solid phase extraction (SPE), due to its limited sensitivity. Recently developed LC–MS/MS methods provide greater sensitivity and selectivity than the GC–TEA method [14], [15], [16], [17], [18]. However, sample matrix effects can lead to poor analyte recoveries and decreased accuracy and precision. Sample clean-up using L–L or SPE [15], [18] can reduce these effects, but this increases analysis time and limits sample throughput. Another method uses stable isotopically labeled standards [15], [16], [17]. Although this approach is effective, its significantly increased cost makes it impractical for routine use. Recently, Zhou et al. [19] described a quantitative and sensitive method for the determination of four TSNAs (NNN, NNK, N-nitrosoanatabine (NAT) and N-nitrosoanabasine (NAB)) in mainstream cigarette smoke using gas chromatography coupled to ion-trap tandem mass spectrometry. Despite the high specificity of ion-trap (IT)-MSn, a drawback of the method is the need for sample preparation by L–L extraction, pre-concentration, and SPE clean-up.

Here we describe a new analytical approach for determination of TSNAs in SHS samples. The method takes full advantage of the specificity and sensitivity of IT-MSn, so that sample preparation involves only extraction of SHS-loaded substrates with methanol followed by centrifugation. We illustrate the method by measuring concentrations of NNN, 4-(methylnitrosamino)-4-(3-pyridyl) butanal (NNA), NNK and NNAL in actual SHS samples. Coupling the high sensitivity/specificity of an ion-trap with minimal sample preparation results in a simple assay that greatly increases sample throughput compared with existing methods for determination of TSNAs.

Section snippets

Caution

NNN, NNA, NNK and NNAL are carcinogenic and must be handled with extreme care inside a fume hood with ventilation using appropriate personal protective equipment (nitrile gloves, lab coat, safety glasses).

Chemicals

NNN, NNA, NNK and NNAL were from Toronto Research Chemicals (Ontario, Canada). Reagent-grade nicotine and quinoline (internal standard, IS) were from Aldrich Chemicals (Milwaukee, WI). HPLC grade methanol and acetonitrile were from Burdick & Jackson (Muskegon, MI).

Standard and QC sample preparation

Stock solutions of nicotine

Results and discussion

Our goal was to develop and validate a simple and rapid method for the detection/identification/quantification of TSNA in SHS samples. To achieve maximum sensitivity/selectivity, the method development was carried out following a three step process: (i) selection of appropriate precursor/product ions and elucidation of fragmentation patterns; (ii) optimization of the IT-MS/MS parameters; (iii) evaluation of the method for the analysis of real SHS samples.

Conclusions

We have developed and validated a sensitive, selective and fast GC–IT-MS/MS method that provides a powerful analytical tool for unequivocal identification and reliable measurement of TSNAs in secondhand tobacco smoke samples. MS/MS parameters, ion structures and fragmentation pathways were investigated to achieve sufficient sensitivity and specificity and thus avoid the use of cleanup procedures.

Analysis of TSNAs in SHS samples collected from a simulated but realistic indoor environment

Acknowledgments

The authors thank A.T. Hodgson (Berkeley Analytical Assoc.) and R. Goth-Goldstein (LBNL) for their helpful comments on the manuscript, Prof. J.F. Pankow (Portland State Univ.) and P. Jacob III (University of California San Francisco) for helpful discussions, and M. L. Russell, F. Mizbani and Y. Carrasco for laboratory assistance. Experimental work was carried out at LBNL under U.S. DOE Contract DE-AC02-05CH11231. This project was supported by the University of California Tobacco-Related

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