A liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify nicotine in gas vapour phase (GVP) collected in calcium and magnesium free phosphate buffered saline (CMF-PBS). In this method the aerosol was trapped in impingers containing ice cold CMF-PBS. An aliquot of the solution was then diluted with a solution containing pyridine as internal standard. The method has been shown to be accurate, precise and linear with a high coefficient of determination (R² > 0.9993).

The Limits of Detection (LOD) as well as of Quantification (LOQ) are 0.08 µg/mL. The initial objective was to achieve a LOQ of 0.05 µg/mL, however this was not feasible, as the variability of the nicotine levels in the blanks (with internal standard) was too high. The working range of the method was from 0.08 up to 50 µg/mL. The inter-run precision (n = 15) is ≤ 10 %; the inter run accuracy (n = 15) for is < ±10 %. The intra run-precision (n = 5) is ≤ 20 %; the intra run accuracy (n = 5) is < ±20 %.

Sensitivity of the method was assessed by dilution experiments; i.e. samples were ten-fold diluted. Five replicates from two samples were analysed for sensitivity. The determined average concentrations of the diluted samples were within the acceptance criteria of < ±15 % of the theoretical concentration.

A stability assessment was performed for both, the refrigerated (2-8 °C) and the frozen (20 °C) samples. QC samples (n = 5) were analysed at time point ‘0’ (study begin) and at the end of the study (after 18 days). All analysed samples, i.e. under each storage condition as well as at each point in time, passed the stability criteria of < ±15 % for accuracy and the %RSD.

This method was developed in order to support the characterisation of GVP samples for in-vitro testing. To ensure proper air-liquid interface exposure during sampling, additional solvent traps were used during sample generation.