Screening for Unknown Substances in Nicotine Pouches

Screening for unknown substances contained in nicotine pouches was performed in part 1 of this study where the procedure is described in more detail (Mallock-Ohnesorg et al. 2023 (link)). In brief, a method using liquid–liquid extraction (LLE) and gas chromatography with mass spectrometric detection (GC/MS) was adapted from Hutzler et al. (2014 (link)). Nicotine pouches were submersed in ultra-pure water and extracted with ethyl acetate under acidic conditions (after addition of 0.1 M hydrochloric acid) and basic conditions (after addition of 0.2 M ammonia). A 2 µl aliquot of the organic phase was injected into the GC/MS system and separated on a DB-17 ms capillary column (30 m × 0.25 mm I.D., 0.25 µm film thickness; Agilent Technologies, Waldbronn, Germany). Peaks were identified using the software Mass Hunter Qualitative Analysis version 10.0 (Agilent Technologies, Waldbronn, Germany) and MSD ChemStation version F.01.03.2365 (Agilent, Technologies, Waldbronn, Germany) and three different spectra libraries: NIST spectral library version 11, Flavor & Fragrance Natural & Synthetic Compounds 3 (FFNSC3) library, and an in-house aroma library created with solutions of standard substances. Nicotine was included as a reference to calculate relative retention times (RRTs). For substances that were included in the in-house library, identification was verified using the RRTs (± 0.05).