SUMMARYThe objective of this study was to develop and validate an analytical method for determining nicotine and nicotine related compounds (i.e., nicotine-N-oxide, cotinine, nornicotine, anatabine, myosmine, anabasine, and β-nicotyrine) in e-cigarette aerosols and e-liquids. Aerosol collection was achieved using a Cambridge collection pad. The sample preparation consisted of adding deuterated internal standards to the collection pad and extracting with 100 mM ammonium acetate solution using a wrist-action shaker. The filtrate was then analyzed by LC-MS/MS using a Gemini NX C 18 column (3 µm, 150 × 3 mm) with a mobile phase gradient system consisting of acetonitrile and 10% acetonitrile in 10 mM ammonium bicarbonate (pH = 8.0) and electrospray ionization (ESI) in the positive mode. The e-liquid was analyzed using the same instrumental parameters, but simplifying the sample preparation procedure by adding deuterated internal standards directly to the 100-mg sample. The sample was then extracted with 100 mM ammonium acetate solution, sonicated, and filtered. In this study, the method's accuracy, robustness, and reliability were enhanced by using deuterated analogues of each compound as internal standards and by applying two ion-transition pairs for each compound for the confirmation and quantification. Validation experiments demonstrated good sensitivity, specificity and reproducibility. All the target compound calibrations exhibited satisfactory linearity from 0.050 to 5.0 mg/mL (r 2 > 0.995). The average recoveries for e-liquids varied from 85.2%(nicotine-N-oxide) to 110% (β-nicotyrine) with recoveries for all compounds exhibiting a coefficient of variation (CV) < 5.0%. Similarly, the average recoveries for e-cigarette aerosols varied from 87.8% (for nicotine-N-oxide) to 111% (for myosmine) with all CV < 8.8%. The LOD and LOQ for e-liquids for all target compounds ranged from 0.234 and 0.781 μg/g (cotinine) to 1.66 and 5.48 μg/g (nicotine-Noxide). For e-cigarette aerosols these limits ranged from 0.094 and 0.312 μg/collection (cotinine) to 0.872 and 2.87 μg/collection (nicotine-N-oxide). This methodology was used to quantitatively determine if any of the target compounds were present in a variety of sample matrices, including e-cigarette solutions and aerosols, and was successfully applied to stability studies, to monitor changes in the target compound levels which might be caused by e-cigarette formulations, components and the storage conditions. [Beitr. Tabakforsch. Int. 27 (2017)