Vanquish duo uhplc system

Samples were reconstituted in 100 µL LC-MS-grade H₂O and analyzed with reversed phase liquid chromatography coupled to high-resolution Orbitrap mass spectrometry. Waters Acquity HSS T3 (2.1 × 150 mm, 1.8 µm) column was used, mobile phase A was H₂O (0.1% formic acid), and mobile phase B was 100% methanol. The following gradient was used at a flow rate of 0.3 mL min⁻¹ and 40 °C: 0.0–2.0 min 0% B, 2–6 min 0–40% B, 6–8 min 40–100% B, 8–11 min 100% B, and at 11.1 min switch to 0% B, 11.1–15 min 0% B. The injection volume was 10 µL. The Vanquish Duo UHPLC-system (Thermo Fisher Scientific) was used.
High-resolution mass spectrometry was done with a high field Thermo Scientific™ Q Exactive HF™ quadrupole-Orbitrap mass spectrometer equipped with an electrospray source. The ESI source parameters were the following: sheath gas 40, auxiliary gas 3, spray voltage 2.8 kV in negative and 3.5 kV in the positive mode, capillary temperature 280 °C, S-Lens RF level 30 and auxiliary gas heater 320 °C. Spectral data were acquired in profile mode. Resolution = 60.000, mass range = 60–900 m/z, AGC target 10⁶, both in positive and negative polarities. Quantification was done through the areas of extracted ion chromatograms of [M+H]⁺ and [M-H]⁻ with 5 ppm mass tolerance on the U¹²C to U¹³C ratio.

Liquid chromatography was performed using a C18 Acquity UHPLC HSS T3 reversed phase column (2.1 × 150 mm, 100 Å, 1.8 µm, Waters, Vienna, Austria) equipped with a VanGuard Pre-column (2.1 × 5 mm, 100 Å, 1.8 µm, Waters, Vienna, Austria) at a column temperature of 40 °C. The flow rate was 0.25 mL/min and the backpressure was 460 bar at the starting conditions. Gradient elution with a total runtime of 30 min was performed using the solvent A: ACN:H₂O (3:2, v/v) and the solvent B: IPA:ACN (9:1, v/v), both of which contained 0.1% formic acid and 10 mM ammonium formate.
The gradient can be described as follows: 0–2 min 30% B, 2–3 min ramp to 55% B, 3–17 min ramp to 67% B, 17–22 min ramp to 100% B, 22–26 min 100% B, followed by an equilibration step from 26 to 30 min using 30% B. A Vanquish Duo UHPLC system (Thermo Fisher Scientific, Germering, Germany) was used and injections were performed with an autosampler. An injection volume of 10 µL was chosen and the injector needle was flushed with 75% IPA and 1% formic acid in between the injections.

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.