Validation of Extraction Method for Tobacco-Free Nicotine Pouches and Swedish Snus Pouch Products Towards Standardization of In Vitro Testing

      Talikka, M.; Goralczyk, A.; Dos Santos, D.; Alriquet, M.; Zanetti, F.; Ortega Torres, L.; Pak, C.; Berthouzoz, M.

      Conference date
      Oct 10, 2023
      Conference name
      CORESTA Smoke Science and Product Technology 2023

      Tobacco-free nicotine pouches (NP) and tobacco-containing Swedish snus pouches (Swedish snus) are two types of nicotine-delivery products for oral use. A standard method to generate an extract of these products for in vitro testing has not been established. Here, we describe the development and validation of an extraction method for NP. Four NPs with various flavours and nicotine contents were tested using five commonly used reference solvents: phosphate-buffered saline, Dulbecco’s Modified Eagle’s Medium, RPMI-1640 Media, McCoy’s 5A Glutamax Media, and artificial saliva. Extractions were performed at 37 °C to mimic the human oral cavity. Pouches were cut in half and infused in the solvents at a 1/5 w/v dilution under gentle shaking (400 min-1) for 1 h. The extracts were centrifuged, and the supernatant was filtered through 0.45 μm and 0.22 μm pore filters.

      Extraction selectivity was confirmed by quantifying nicotine and menthol levels and qualifying the presence or absence of other target flavour-related ingredients. The coefficient of variation (CV) between runs reached a maximum of 9 % for menthol (as a representative flavour) and 2 % for nicotine quantification, confirming good intermediate precision (i.e., the extraction method was not impacted by variables such as the instrument, day of analysis, or operator). Nicotine extraction recovery was ~100 % (CV 5 %) across all solvents and pouches. The effect of time on extraction efficiency was evaluated by extracting the pouches for 1 h ± 20 %. A maximum difference of 6 % compared to reference timing was measured, indicating the variation was linked to the method itself rather than the impact of the infusion timing. Stability was also evaluated for two months at -80 °C storage and indicated no loss of nicotine in the trapped solvents. In conclusion, we successfully developed and validated an extraction method for NP that can be used as a standard for future in vitro assessment.