Nicotine concentrations were determined by LC-MS using the Agilent 1260 Infinity liquid chromatography system with 6130 quadrupole MS. Aliquots (20 μl) of each sample were injected into a Poroshell 120 EC-C8 column (4.6 mm by 50 mm, 2.7 μm; Agilent Technologies) and subjected to a gradient (A to B, where A = 0.1% formic acid in water and B = 0.1% formic acid in acetonitrile) of 0% B for 3 min, 0% B to 100% B from 3 to 7 min, and 100% B from 7 to 10 min at a constant flow rate of 0.5 ml/min. A column-solvent equilibration time of 3 min was implemented before the next sample was analyzed. The following were the MS operational parameters: API-ES (atmospheric pressure ionization electrospray) mode, channel 1 (90%) positive single ion monitoring of mass/charge ratios 166 (50%, nicotine D3) and 163 (50%, nicotine) and channel 2 (10%) scan for positive ions, nitrogen as a nebulizing and drying gas (35 psi, 12 L/min), high capillary voltage of 4 kV, and drying gas temperature to 300°C. To protect the detector from salts in the buffer, MS was turned on with a delay of 1.4 min after the injection.

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