Dilute and Shoot Method for the Determination of Tobacco-Specific Nitrosamines (TSNAs) in Smokeless Tobacco Products by UPLC-MS/MS

Significance of the Topic

• TSNAs are potent carcinogens formed during tobacco processing and curing.
• Regulatory bodies mandate accurate quantification of these nitrosamines in tobacco products and emissions.
• A robust analytical approach supports tobacco harm reduction and quality control.

Objectives and Study Overview

The study aims to develop and validate a rapid 'dilute and shoot' UPLC-MS/MS procedure for detecting four major tobacco-specific nitrosamines (NNN, NNK, NAT, NAB) across diverse smokeless tobacco matrices. It compares the performance against established CORESTA/ISO methods and explores improvements in sensitivity, throughput, and workflow simplicity.

Methodology

1 g of tobacco was extracted with 100 mM ammonium acetate in water, shaken for 30 min, filtered, and diluted tenfold. Chromatographic separation was carried out on a 2.1 × 50 mm, 1.7 μm BEH C18 column at 45 °C with a 0.45 mL/min flow, using a water-based ammonium acetate mobile phase and 0.1 % acetic acid in methanol. Tandem MS detection in positive ESI mode employed MRM transitions optimized via IntelliStart. RADAR acquisition enabled simultaneous full-scan MS and MRM to assess matrix effects and optimize resolution. Isotopically labeled internal standards (NNN-D4, NNK-D4) ensured accurate quantification.

Instrumentation Used

• ACQUITY UPLC H-Class system
• ACQUITY UPLC BEH C18, 2.1 × 50 mm, 1.7 μm column
• Xevo TQD tandem quadrupole mass spectrometer
• MassLynx and TargetLynx data processing software

Key Results and Discussion

Calibration curves for all four TSNAs were linear over a wide range (R² > 0.999). A 0.05 ng/mL standard yielded signal-to-noise ratios >18 for all analytes, demonstrating high sensitivity. Recoveries from spiked tobacco matrices ranged from 105 % to 119 % (RSD ≤ 7 %), indicating accurate quantification without solid-phase extraction. UPLC-MS/MS data matched inter-laboratory HPLC-MS/MS results on CORESTA reference products, confirming method robustness and minimal matrix interference.

Benefits and Practical Applications

• Rapid 7-minute analysis increases sample throughput.
• Dilute-and-shoot workflow removes the need for SPE cleanup, reducing preparation time and instrument maintenance.
• Extended dynamic range and low detection limits support diverse tobacco matrix analysis with a single protocol.
• RADAR-based method optimization accelerates development and mitigates matrix suppression.

Future Trends and Potential Applications

Future developments may include high-resolution mass spectrometry for broader nitrosamine screening, automated sample handling, and application to heated tobacco and e-vapor products. Continued method refinement will address evolving regulatory requirements and expand the analyte scope to other harmful constituents.

Conclusion

The UPLC-MS/MS 'dilute and shoot' approach provides a sensitive, fast, and streamlined alternative for TSNA quantification in a range of tobacco products. By enhancing chromatographic resolution, extending calibration limits, and eliminating extensive cleanup, the method facilitates regulatory compliance, quality control, and research in tobacco harm reduction.

References

1. FDA Guidance for the Industry. Reporting Harmful and Potentially Harmful Constituents in Tobacco Products and Tobacco Smoke Under Section 904(a)(3) of the Federal Food, Drug, and Cosmetic Act. March 2012.
2. CORESTA Recommended Method No. 63. Determination of Tobacco Specific Nitrosamines in Cigarette Mainstream Smoke by GC-TEA. 2005.
3. CORESTA Recommended Method No. 72. Determination of Tobacco-Specific Nitrosamines in Smokeless Tobacco Products by LC-MS/MS. July 2013.
4. ISO 22303: Tobacco – Determination of Tobacco-Specific Nitrosamines – Method Using Buffer Extraction. 2008.
5. CORESTA STS Technical Report: Smokeless Reference Tobacco Product Analysis. 2014.