Aquity uplc system

For UPLC-qTOF, 120 μl of the resolved extract was transferred to an autosampler vial for analysis. Chromatographic separation was performed using an AQUITY UPLC system (Waters), equipped with a BEH-C18-column (2.1 × 150 mm, 1.7 μm; Waters) as previously described [150 (link)]. A SYNAPTG1 qTOF HD mass spectrometer (Waters) equipped with an ESI source was used for detection. For positive and negative ionization mode, 5 μl and 10 μl were injected, respectively. Data acquisition was done by the MassLynx 4.1 software (Waters Corporation). Lipid classes were analyzed with the “Lipid Data Analyzer 1.6.2” software [151 (link)]. Extraction efficacy and lipid recovery were normalized using ISTD, and lipid classes were expressed as percent composition.

Total lipids of lung explants (5–30 mg) were extracted twice according to Folch et al. (68 (link)) using chloroform/methanol/water (2/1/0.6, vol/vol/vol) containing 500 nM butylated hydroxytoluene, 1% (vol/vol) acetic acid, and 4 nmol of internal standard mix (C17-lysophosphatidylcholine, C17-phosphatidylcholine, C17-triacylglycerol) per sample. Extraction was performed under constant shaking for 60 minutes at room temperature. The organic phase was collected after centrifugation (1000g for 15 minutes at room temperature). Combined organic phases of the double extraction were dried under a stream of nitrogen and dissolved in 200 μL of chloroform/methanol/2-propanol (2/1/12, vol/vol/vol) for UPLC-qTOF analysis. Chromatographic separation was performed using an AQUITY-UPLC system (Waters Corp.), equipped with an HSS T3 column (2.1 × 100 mm, 1.8 μm) as previously described (67 (link)). A SYNAPTG1 qTOF HD mass spectrometer (Waters Corporation) equipped with an ESI source was used for detection. Data acquisition was done with MassLynx, version 4.1, software (Waters Corp.). Lipid classes were analyzed with Lipid Data Analyzer, version 1.6.2, software (69 (link)). Extraction efficacy and lipid recovery were normalized using internal standards. Lipid profiling was performed in-house.

The quantitative assay of ABA in developing maize embryos was preformed using ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UPLC/ESI-MS/MS) [55 ]. The HPLC system consisted of Aquity UPLC™ System (Waters, Milford, MA, USA) quaternary pump equipped with an autosampler. A Poroshell 120 column (EC-C18, Agilent, 3 × 100 mm, 2.7 μm) was used for ABA separation, with 3 μL injection volume. Gradient elution was done with 0.1% formic acid/H₂O (solvent A) and acetonitrile with 0.1% formic acid/acetonitrile (solvent B) at a constant flow rate of 0.6 mL/min. Mass spectrometry (MS) and MS/MS were done in multiple reaction mode on an API 5500 triple quadrupole mass spectrometer (AB Sciex, Boston, MA, USA), assisted with electrospray ionization (ESI) and Analyst® Software 1.6.2. The optimized conditions were as follows: curtain gas: 30; collision gas: 8; ion spray voltage: 3000 V; temperature: 500 °C; ion source gas 1: 35; gas 2: 45.

5 µL of protein extract was injected into a C18 column, 300 Ǻ, 2.1 mm × 150 mm (Sigma, Saint-Quentin Fallavier, France) maintained at 60 °C. LC-MS was performed with an Aquity UPLC system connected on-line with a Waters Xevo G2-XS-TOF mass spectrometer (Waters, Guyancourt, France). The following gradient was used at a flow rate of 0.2 mL/min: isocratic hold at 5% B for 3 min followed by three steps of linear increases to 25% B at 4 min, 55% B at 34 min, 80% B at 36 min, followed by an isocratic hold at 80% B for 9 min. The column was finally equilibrated with 5% B for 10 min prior to the next run. Solvent A was 99.9% H₂O/0.1% formic acid and solvent B was 90% ACN/9.9% H₂O/0.1% formic acid.
MS source parameters were: ESI capillary voltage +4.3 kV, desolvation temperature 350 °C, cone gas flow rate 10 L/min, nebulizer pressure 25 psig, and fragmentor voltage 250 V. Data were acquired at 1 spectrum/sec, with acquisition window 100 to 3000 m/z.

All reagents used were commercially available. All solvents were of HPLC grade. Analytical LC-MS analysis was performed on a Waters AQUITY UPLC system equipped with PDA and SQD MS detector; column: AQUITY UPLC BEH C18 1.7μm, 2.1 x 50mm; column temp: 65°C; solvent A: 0.1% formic acid (aq); solvent B: 0.1% formic acid (acetonitrile); gradient: 5% B to 100% B in 2.4 min, hold for 0.1 min, total run-time ca. 2.6 min. ¹H and ¹³C NMR 300 MHz spectra were recorded on a Varian Mercury 300 BB spectrometer at room temperature. All NMR spectra were recorded using CDCl₃ or DMSO-d₆ as solvents

Phytochelatins (PCs) were determined by UPLC-ESI-QTOF-MS as described earlier^{53 (link)}. Briefly, frozen homogenized plant material was extracted with 0.1% (v/v) trifluoroacetic acid containing 6.3 mM diethylene triamine pentaacetic acid and 40.04 µM N-acetylcysteine as internal standard. Prior to derivatization with monobromobimane at 45 °C for 30 min, thiol groups were reduced with Tris-(2-carboxyethyl)-phosphine. Labelled thiols were separated on a HSS T3 column (1.8 μm, 2.1 × 100 mm; Waters Corporation, Milford, MA, USA) by a Waters Aquity UPLC system applying a linear binary gradient of water (A) and acetonitrile (B), both acidified with 0.1% (v/v) formic acid, at a flow of 0.5 mL min^–1: 99.5% A, 0.5% B for 1 min, a linear gradient to 60.5% B at 10 min, gradient to 99.5% B at 12 min, flushing with 99.5% B for 1 min, a gradient back to initial conditions in 1 min and an additional re-equilibration for 1 min. The column temperature was set to 40 °C. Thiols were detected with a Q-TOF Premier mass spectrometer equipped with an ESI-source (Waters Corporation) operated in the V + mode. For quantification the QuanLynx module of the MarkerLynx software was used.