Introduction - The method mainly used for the determination of N-nitrososarcosine (NSAR) in tobacco – gas chromatography coupled with a thermal energy analyzer – lacks selectivity and requires derivatization and laborious sample cleanup. A different technique, liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), is advanced but involves difficulties regarding (i) the retardation of the polar compound NSAR and (ii) unequal ESI-MS/MS responses of the syn and anti conformers of NSAR.

Experimental - LC-ESI-MS/MS measurements were carried out using an Obelisc N column in Hydrophilic Interaction Liquid Chromatography (HILIC) mode coupled with a triple quadrupole MS in negative ionization mode. Samples were extracted with aqueous formic acid and subsequently purified with solid supported liquid-liquid extraction. For quantification external calibration with internal standard-correction using the isotope labeled NSAR-D3 was applied. Nuclear magnetic resonance spectroscopy was used to assign the syn and anti conformers and to determine their ratios.

Results and Discussion - The selected stationary phase in HILIC mode is capable of retaining the polar compound NSAR reasonably. Using two different LC gradient methods it was observed that the conformers show different ESI-MS/MS response and that their ratios differ in real samples and in standards of different age. As a consequence, for proper quantification the conformer ratio in the external standard needs to be adjusted to the same ratio as in the samples.

The method described has been applied to several tobacco products and has been fully validated for CORESTA Reference Product 2 (moist snuff) and 3 (dry snuff) showing expanded measurement uncertainties of approximately 20% and limits of quantification of about 20 ng/g.