Determination of nicotine and nicotine metabolites in urine by hydrophilic interaction chromatography–tandem mass spectrometry: Potential use of smokeless tobacco products by ice hockey players
Introduction
Nicotine is the principal natural alkaloid present in tobacco leaves. A wide variety of consumption patterns exist, from tobacco smoking, in the form of cigarettes, cigars or pipes, to smokeless tobacco products such as snus, snuff and chewing tobacco. Nicotine replacement therapies also contain this natural compound, as marketed in transdermal patches, nasal sprays, inhalers and gums [1].
Depending on the type of product, concentrations differ to a reasonable extent. On average, a similar content of nicotine is found in cigarette and oral snuff, whereas cigar and chewing tobacco contain only about half of this concentration [1]. Accordingly, levels of nicotine intakes and metabolism pathways vary along these different trends of tobacco consumption. When smoked and inhaled, nicotine is rapidly absorbed in the lungs, reaching the brain via the bloodstream within 20 s [1]. Depending on the pH, there is little to large buccal absorption, which is directly related to the type of product [2], [3]. Chewing tobacco and snus are buffered to facilitate absorption of nicotine through oral mucosa. A portion of nicotine is usually swallowed with saliva and well absorbed in the small intestine. Concentration in the brain rises at a slower rate than with smoking and levels are declining over a longer period of time. Nicotine is also well absorbed through the skin which is the basis for transdermal delivery that occurs over a long period of time [4].
Nicotine is primarily and extensively metabolized in the liver by C-oxidation to cotinine [2], [5]. N-oxidation also converts nicotine into nicotine-N′-oxide and other minor metabolites. Cotinine is further hydroxylated to trans-3-hydroxycotinine and also converted to cotinine-N-oxide and other minor metabolites by N-oxidation (Fig. 1). Simultaneous determination of free urinary nicotine, cotinine, trans-3-hydroxycotinine, nicotine-N′-oxide and cotinine-N-oxide account for 8–10, 10–15, 33–40, 4–7 and 2–5% of the total nicotine dose, respectively [5], [6].
Due to the relatively short half-life of nicotine in urine (about 2 h), investigating nicotine metabolites which exhibit a longer half-life is a prerequisite to provide relevant information on tobacco consumption [1]. Therefore, an abundant literature on gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods has been published for the determination and quantification of nicotine and selected metabolites in biological fluids, including blood or plasma, urine and saliva [6], [7], [8], [9], [10], [11], [12], [13], [14].
LC–MS/MS provides a sensitive and selective approach for comprehensive measurement of free nicotine and its metabolites. However, only very few of these publications bring up a simultaneous, yet steps limited, sample preparation method for the analysis of nicotine and metabolites, in particular nicotine-N′-oxide and cotinine-N-oxide [6], [9], [10], [12].
Nicotine can act both as a stimulant and a relaxant drug, with predominant effects being an increase in pulse rate and blood pressure, as well as an increase of blood sugar release and the release of epinephrine [4], [15]. Positive reinforcing effects also include relaxation, reduced stress, enhanced vigilance, improved cognitive function, mood modulation and lower body weight [3], [16].
Thus, when considering nicotine from a doping perspective, consumption in the form of smokeless nicotine products may clearly enhance the performances of sport athletes in various ways as it provides all the effects described above, without the direct health issues usually associated to smoke [17]. Indeed, use of snus, snuff or chewing tobacco has been reported as a growing trend, in particular amongst winter sports such as ice hockey and skiing, but also in other popular sports such as soccer, baseball or basketball and even in fencing or shooting [18], [19], [20]. Nevertheless, only old and vague estimates of these consumption patterns have been reported, leading to an extensive underestimate of this potential issue. Nicotine did not appear in the 2009 World Anti-Doping Agency (WADA) Prohibited List or in the 2009 Monitoring Program, a situation which remains unchanged at the present time [21], [22]. Thus, investigating nicotine consumption trends amongst professional athletes and developing means to distinguish between consumption of smoke or smokeless nicotine products is of major concern to sport authorities.
Therefore, the project presented in this paper describes an analytical method for the simultaneous determination and quantification of nicotine and its four main metabolites in urine, using liquid–liquid extraction (LLE) followed by liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI–MS/MS) in Hydrophilic Interaction Chromatography (HILIC) mode. Apart from a recent publication on nicotine, cotinine and trans-3-hydroxycotinine analysis [23], HILIC columns have never been used for such purpose in real biological samples, in particular when including nicotine-N′-oxide and cotinine-N-oxide. However, this methodology is primarily dedicated to the analysis of polar compounds, such as molecules and related metabolites excreted in urine [24], [25]. Owing to the nature of screening procedures for doping agents, a rapid and simple extraction procedure is favoured for comprehensive nicotine consumption study.
This analytical approach has been further applied to the urine samples collected during the 2009 Ice Hockey World Championships held in Switzerland in order to measure the prevalence of nicotine exposure amongst athletes and help to assess the concern associated with nicotine consumption in sport.
Section snippets
Reagents and chemicals
(S)-Nicotine (≥99%) and (S)-cotinine (98%) were purchased from Sigma-Aldrich (Buchs, Switzerland), trans-3-hydroxycotinine (99.9%), (R/S)-nicotine-N′-oxide (98%) and (S)-cotinine-N-oxide (98%) were obtained from Toronto Research Chemicals (Toronto, Canada), while (S)-d4-nicotine (98.8%), (R/S)-d3-cotinine (99%) and d3-trans-3-hydroxycotinine (98%) were supplied by LGC Promochem (Molsheim, France). Acetonitrile (ACN, ≥99.7%) was purchased from Biosolve B.V. (Chemie Brunschwig, Basel,
LC–MS/MS analyses
A complete separation of nicotine and metabolites in urine specimens was achieved by hydrophilic interaction chromatography using a gradient of ACN (A) and 10 mM ammonium formate (pH 3.0) buffer (B) with a flow rate set at 0.3 mL/min (Fig. 2). Indeed, HILIC mode allowed to successfully isolate each analyte by providing adequate retention of polar compounds and excellent peak shape. Sensitivity was also optimized since using a mobile phase highly enriched in polar organic solvent ensures an
Conclusion
A sensitive and selective HILIC–ESI-MS/MS method for the simultaneous detection and quantification of nicotine and its four principal metabolites in urine was developed and fully validated. The simple and fast sample preparation protocol based on LLE provided a satisfactory matrix clean-up and recovery, while the subsequent use of hydrophilic interaction chromatography allowed to obtain very good separation and peak shape, enhanced sensitivity and high samples throughput.
This analytical
Acknowledgements
This project was entirely supported by a grant from ADS. The authors would also like to gratefully acknowledge the IIHF for making this study possible.
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