Robust and Fast Analysis of Four Tobacco Specific Nitrosamines in Tobacco Cigarettes by LC–MS-MS Using RSLC PA2 Column

Tobacco Specific Nitrosamines (TSNA) are a group of carcinogens found only in tobacco products and are formed from nicotine and related alkaloids during the production and processing of tobacco and tobacco products (1). Due to their carcinogenic properties, efforts have been made to reduce TSNA levels in tobacco products. The project goal was to demonstrate a high throughput and sensitive method to monitor TSNA levels.

Conventional methods for TSNA analysis are based on gas chromatography with thermo energy analyzer (GC-TEA) (2,3,) or high performance liquid chromatography (HPLC) with various detection techniques, such as UV and mass spectrometry (MS) (4-6). This application note describes a robust and fast LC–MS-MS method for the determination of four TSNAs in tobacco cigarettes.

Figure 1

Experimental

Chromatographic Conditions

Analytical Column: Acclaim® RSLC PA2 (5.0 × 2.1 mm, 2.2 μm)

Column Temperature: 60 °C

Injection Volume: 10 μL

Mobile Phase: 10% CH₃CN in buffer (1 mM NH₄OAc, pH adjusted to pH 8.0 by NH₄OH)

Flow Rate: 0.50 mL/min

Detection: UV @ 230 nm TSQ Quantum Access™ SRM with H-ESI

Mass Spectrometric Conditions

See Figure 1.

The first SRM transition of each compound is used for quantification and the second SRM transition is used for confirmation.

NNN: N'-nitrosonornicotine, NAT: N'-nitrosoanatabine, NAB: N-nitrosoanabasine. NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Method Performance

Figure 1 shows the four TSNAs retained and completely resolved within 3.5 min, with the minimum of the retention factors (K'NNN) being greater than four and minimum resolution (RsNAT) greater than two. Demonstrated retention factors ensure the chromatographic separation of analytes of interest from early eluted compounds, which minimizes the possibility of ionization suppression and eliminates cross contamination of SRM transitions between main analytes. The use of specific MS-MS detection allows the simplification of sample preparation, and the greater sensitivity ensures quantification confidence at low levels.

Since the analytical column was running under the very harsh conditions of high temperature and high pH mobile phase, method ruggedness was evaluated by repeated injections of standard solutions and tobacco extracts and was well maintained after more than 1000 injections.

Conclusion

A simple, sensitive, and specific method for the determination of tobacco specific nitrosamines is demonstrated and has been applied to the analysis of TSNAs in different cigarettes.

References

(1) Hecht, S. S.; Hoffmann, D. Carcinogenesis 1988, 9, 875–884.

(2) Adams, J. D.; Brunnemann, K. D.; Hoffmann, D. J.Chromatog.,A 1983, 256, 347–351.

(3) Krull, I. S.; Swartz, M.; Hilliard, R.; Xie, K. H.; Driscoll, J. N. J. Chromatog., A 1983, 260, 347–362.

(4) Wu, W.; Ashley, D. L.; Watson, C. H. Anal. Chem. 2003, 75, 4827–4832.

(5) Wagner, K. A.; Finkel, N. H.; Fossett, J. E.; Gillman, I. G. Anal. Chem. 2005, 77, 1001–1006.

(6) Wu, J.; Joza, P.; Sharifi, M.; Rickert, W. S.; Lauterbach, J. H. Anal. Chem. 2008, 80, 1341–1345.

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