Orbitrap idx mass spectrometer

Simultaneous determination of nine alkaloids (nicotine, anatabine, anabasine, myosmine, nicotinamide, cotinine, nornicotine, norcotinine, and nicotinic acid) and their respective deuterated or ¹³C internal standards (IS) was performed on a Vanquish Duo UHPLC system coupled to an Orbitrap IDX mass spectrometer (Thermo Fisher Scientific) operating in positive electrospray ionization mode scanning the 50–800 amu mass range at 60k resolution. Chromatographic separation of the injected sample (3 µL) was achieved on an Acquity HSS T3 column, (150 × 2.1 mm, 1.7 µm from Waters, Milford, MA); the column temperature was set to 45 °C. Ammonium acetate in water (10 mM; pH 8.9; mobile phase A) and ammonium acetate in methanol (10 mM; mobile phase B) were applied as a gradient (0–0.25 min: 10% B; 4.25 min: 98% B; 5.5 min: 98% B, 5.6 min: 10% B until 7 min) with a constant flow rate of 0.3 mL/min.

Additional details of the study procedures, including laboratory assays, questionnaires, and food record completion have been previously published in a manuscript describing the rationale and design of the study [26 (link)]. Liquid chromatography-mass spectrometry (LC-MS) instruments were upgraded to a Thermo Scientific Vanquish UPLC coupled with an Orbitrap ID-X mass spectrometer (Thermo Scientific, Waltham, MA, USA), performed in negative ion mode. A Waters Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µm) was used and maintained at 35 °C. Mobile phase containing solvent A (H₂O with 0.1% NH₃) and B (ACN with 0.1% NH₃) at a flow rate of 300 µL/min, d₆-sucralose and d₄-ace-K were used as internal standards. Calibration exceeded FDA LC-MS guidelines for linearity and quantitation.