Altis triple quadrupole mass spectrometer

High-performance liquid chromatography (HPLC) grade solvents and LC–MS modifiers were purchased from Sigma-Aldrich (St. Louis, MO, USA). Detection and quantification were achieved by ultra-performance liquid chromatography—tandem mass spectrometry (UPLC-MS/MS) utilizing a Thermo Scientific Vanquish UPLC with a Thermo Scientific Altis triple quadrupole mass spectrometer, heated electrospray ionization (HESI-II) in positive ion mode (3500 V). 50 µl of sample was mixed with 200 µl of acetonitrile (ACN), vortexed for 5 min and then centrifuged at 4 °C, 17,000 g for 15 min. The supernatant was transferred to an LC–MS vial for analysis. Injection volume was 1 µl. A Waters Cortecs T3 column, 2.1 × 100 mm, 1.6 µm column was maintained at 35 °C. Solvent A: H₂O with 0.1% formic acid (FA) and Solvent B: ACN with 0.1% FA. The flow rate was 250 µl/min, the gradient was 25% B at 0 min for 0.25 min, increasing to 65% B at 5 min, further increasing to 90% B at 5.5 min, remained 90% B until 7.5 min, then decreased to 25% B at 8 min. The total running time was 10 min. Samples were analyzed in triplicates. Quantitation of ¹⁴NAT and ¹⁵NAT were based on multiple reaction monitoring (MRM) transitions m/z, 170.062 → 115.042 and 171.062 → 115.042, respectively. The result was based on the percentage ratio of ¹⁵NAT/(¹⁴NAT + ¹⁵NAT).

Analysis was conducted using a Vanquish LC system, coupled with an Altis triple‐quadrupole mass spectrometer (Thermo, Hemel Hempstead, UK). The mobile phases and column were as detailed above for HRAM analysis, however a different solvent gradient programme was employed: Chromatographic conditions were held with a flow rate of 0.5 ml/min at 98% A for the first 0.5 min, increased to 25% B over the next 3.5 min and then increased to 95% B and the conditions held until 5 min, after which time the system was returned to starting conditions and re‐equilibrated.
The mass spectrometer was operated in positive electrospray ionisation mode with a vaporiser temperature of 400°C and ion transfer tube temperature of 350°C. The spray voltage was 3500 V. Collision energies were set at 26 eV for the fragmentation of higenamine and the deuterated IS, for the SRM transitions m/z 272 > 107 and 276 > 108 for higenamine and the IS, respectively. Calibration lines (analyte/IS peak area vs analyte concentration) were constructed using Thermo XCalibur version 4.0.27.21 QuanBrowser using a weighted linear regression.