Quattro micro api mass spectrometer

The analysis was performed using a Waters ACQUITY UPLC system (Waters, Milford, MA, United States) and a Micromass Quattro Micro API mass spectrometer (Waters, Milford, MA, United States). The electrospray ionization (ESI) source interface was operated in the positive and negative ionization modes in our study. The following parameters were used: capillary voltage: 3.1 kV, source temperature: 150°C, desolvation temperature: 400°C. Nitrogen was used as the desolvation and cone gas at a flow rate of 800 L h⁻¹ and 50 L h⁻¹, respectively. Argon was used as collision gas at a flow rate of 0.17 ml min⁻¹ in the collision cell. Collision energies and cone voltages were optimized for each analyte individually. The MS/MS parameters for antibacterial drugs and ISs are shown in Table 1.
Chromatographic separation was performed on an ACQUITY UPLC^® BEH C₁₈ Column (2.1 × 100 mm; 1.7 µm). The mobile phase consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) used at a flow rate of 0.3 ml min⁻¹. The gradient elution program was as follows: 0–0.5 min, 10% B; 0.5–1.2 min, 10%–35% B; 1.2–3.5 min, 35%–70% B; 3.5–4.2 min, 70%–90% B; 4.2–5.2 min, 90% B; 5.2–5.5 min, 90%–10% B; and 5.5–6.0 min, 10% B. The column was maintained at 45°C, the autosampler was set at 4°C. and the injection volume was 5 µL.

The IAA and oxIAA levels were determined in leaves from WT and lhy‐10 trees, sampled as described above. For each sample, 20‐mg plant tissue was homogenized in cold 0.05‐M sodium phosphate buffer (pH 7.0), containing 0.025% sodium diethyldithiocarbamate and labeled internal standards (¹³C₆‐IAA and ¹³C₆‐oxIAA). Samples were purified by solid phase extraction using mixed‐mode anion exchange sorbent (Oasis™ MAX cartridge, 1 cc/30 mg; Waters Corp., Milford, MA, USA) and injected onto a reversed‐phase column (BetaMax Neutral; 150 mm × 1 mm; particle size 5 μm; Thermo Fisher Scientific, Waltham, MA, USA) with UniGuard™ column protection (Hypurity advance; 10 mm × 1 mm; 5 μm; Thermo Scientific). Sample analyses were performed by ultraperformance liquid chromatography electrospray tandem mass spectrometry analysis using an Acquity UPLC™ System and a Quattro micro™ API mass spectrometer (Waters Corp.; Novák et al., 2012).

HPLC analysis of the examined phytohormones (GA₃, ABA, JA, and SA) was carried out using an HPLC-ESI-MS/MS (Waters Alliance 2695) system consisting of a micro-mass Quattro micro-API mass spectrometer. The separation of compounds was performed at 30 °C on an Atlantis T3-C18 column with 3 μm particle size, 100 mm length, and 2.1 mm diameter. The mobile phase comprised 0.1% formic acid in water (50%) and 0.1% formic acid in acetonitrile (50%) with an isocratic elution program. The flow rate of the mobile phase was 0.2 mL min^–1 throughout the run, and the injection volume for samples was 5.0 µL. We quantified all phytohormones using multiple reaction monitoring (MRM) in negative mode by applying separate retention time windows. The optimal conditions for the ionization source were as follows: ion spray voltage 3500 V, DE temperature 300 °C, source temperature 120 °C, and collision energy 25 eV. Nitrogen was used as curtain gas with a flow rate of 200 L h⁻¹. To determine the concentration of phytohormones in the extracts, we used an equation derived from the calibration curves (y_JA = 2.312x + 0.1699, R² = 0.9991; y_ABA = 1.24256x + 0.99721, R² = 0.9981; yGA₃ = 3.56112x + 0.169908, R² = 0.9991; y_SA = 10.8929 x + 102.371, R² = 0.9974) of standard compounds (JA: Sigma-99%, SA: Merck-99%, ABA: Sigma-99%, and GA₃: Merck-90%) and represented as ng g⁻¹ FW.