6490 triple quadrupole lc ms

Experiments were performed using an Agilent 6490 Triple Quadrupole LC–MS apparatus. A Waters XBridge Amide column (2.1 mm × 100 mm, 3.5 μm particle size; Waters, Milford, MA, USA) was used for chromatographic separation. The column temperature was 35 °C. Mobile phase A comprised acetonitrile/water (50:50, v/v) containing 15 mM ammonium acetate in water containing 0.2% ammonium hydroxide. Mobile phase B comprised acetonitrile/water (95:5, v/v) containing 15 mM ammonium acetate in water containing 0.2% ammonium hydroxide. The gradient was programmed as follows: 0–10 min, 100% B; 10–23 min, 100–0% B; 23–24 min, 0–100 %B; and 24–30 min, 100% B. The flow rate was 0.3 ml/min, and the injection volume was 5 μl.
The parameters for AJS electrospray ionization MS/MS in positive/negative ion mode were as follows: dry gas: nitrogen; dry gas temperature, 200 °C; dry gas flow rate, 14 l/min; nebulizer, 20 psi; sheath gas: nitrogen; sheath gas temperature, 250 °C; sheath gas rate, 11 l/min; capillary voltage, ± 3000 V and nozzle voltage, ± 1.5 kV. Multiple reaction monitoring was performed using the characteristic precursor-to-product ion transitions, fragmentor voltage (380 V), and collision energies. The polar metabolites were identified based on retention time by using authentic standards and quantified through standard curve samples.

The LC-MS/MS method used was developed by Main et al. (2018) [43 (link)] and was carried out on an Agilent 1290 infinity LC system and a Agilent 6490 triple quadrupole LC-MS. LC was carried out on a Kintex^® C18 column (17 μm particle size, 100 Å pore size) (Phenomenex^® Australia, Lane Cove, NSW, Australia), via a gradient elution of the mobile phases (Solvent B’s gradient was as follows: 0.00 min 55%, 10.00 min 68%, 10.10 min 100%, 15.00 min 100%, 15.10 min 55%) with a flow rate of 0.250 mL/min with a column temperature of 35 °C (a typical chromatogram is shown in Figure 2). After each sample, the mobile phase was left at starting conditions for 2 min for column re-equilibration. Samples were run in triplicate injections of 5 μL. Prior to analysis, multiple reaction monitoring (MRM) ion transitions were set up for each analyte and the internal standard (D5-DAB) (Table 4). Data acquisition occurred in electrospray ionisation (ESI) positive mode, the drying gas temperature was 250 °C at 14 L/min, the sheath gas temperature was 250 °C at 11 L/min, with a nebuliser pressure of 20 psi. The limit of detection and the limit of quantification were determined with a signal to noise ratio of 3.3 and 10 respectively. BMAA was calculated to have a LOD of 0.02 pg/μL and a LOQ of 0.05 pg/μL, while both AEG and 2,4-DAB were calculated to have a LOD of 0.04 pg/μL and a LOQ of 0.13 pg/μL.