Salting-Out-Assisted Liquid-Liquid Extraction Method for the Determination of Nicotine from Oral Traditional and...

In this study, we developed and validated a novel method that allows for the extraction and quantitation of nicotine from a variety of commercially available oral tobacco products including loose and pouched traditional moist smokeless tobacco products, and oral tobacco-derived nicotine (OTDN) lozenges, gums, and pouches. The method employed an extraction technique consisting of salting-out assisted liquid-liquid extraction using sodium hydroxide and acetonitrile in conjunction with ultra-high pressure liquid chromatography coupled to mass spectrometry. Accurate quantitation was obtained using nicotine methyl-d₃ isotopically labeled internal standard. Chromatographic separation of nicotine and nicotine methyl-d₃ internal standard was achieved using a Waters Acquity C18 column (50 mm × 2.1 mm i.d., 2.5 μm) with 10 mM ammonium acetate buffer (pH = 10) and acetonitrile as mobile phase A and B, respectively. Using a gradient elution and a flow rate of 0.4 mL/min for 5 min runtime, nicotine eluted at 1.74 min. The method was validated according to ICH guidelines for all the sample types with an accuracy for nicotine within 89-109%. Repeatability and intermediate precision were both estimated to be ≤7% relative standard deviation (% RSD). This method is applicable for a wide range of traditional moist smokeless and OTDN tobacco products.