6550 q tof mass spectrometer

Metabolic flux analysis was performed on a 6550 Q-TOF mass spectrometer with Dual AJS ESI source (Agilent Corporation, MA, USA) equipped with a 1260 RRLC system (Agilent Co.).
LC separation was conducted under both acidic and basic conditions using a Waters BEH amide column (2.1 × 100 mm, 1.7 μm) at a column temperature of 35 °C. Mobile phase A was 95:5 acetonitrile: water with 10-mM ammonium acetate. Mobile phase B was H₂O with 10-mM ammonium acetate. Ammonium hydroxide (0.05%) and acetic acid (0.05%) were added in basic and acidic LC conditions, respectively.
The injection volume was 5 μL, and the flow rate was 0.25 mL/min. The basic gradient conditions were 0 min, 15% B; 12 min, 40% B; and 15 min, 47% B. The acidic gradient conditions were 0 min, 5% B; 5 min, 30% B; 6 min, 50% B; and 12 min, 50% B. The MS parameters were set as follows: Gas Temp, 200 °C; Drying Gas, 14 l/min; Nebulizer: 35 psig; sheath gas temp, 350 °C; sheath gas flow: 11 l/min; VCap, 3500 V; Nozzle Voltage, 600 V; Fragmentor, 380 V; and Octopole RF, 750 V. The data were acquired in a mass range from 70 to 1000 m/z in negative ion mode using MassHunter workstation (Agilent Co., MA, USA).

SAE was analyzed using Quadrupole time-of-flight liquid chromatography–mass spectrometry (Q-TOF–LC/MS) to detect active compounds. SAE in 70% ethanol was loaded in column chromatography using the Sephadex^® LH-20 column (1.0 × 60 cm) at room temperature and the loaded SAEs were fractionized using chloroform as a mobile phase. Q-TOF–LC/MS was operated using an Agilent Infinity 1290 UHPLC connected to an Agilent Eclipse Plus C-18 column (2.1 × 100 mm, 2.1 μm) coupled to an Agilent 6550 QTOF mass spectrometer^{18 (link)}. The injection amount was 1 μL, and the temperature of the column was maintained at 40 °C. The mobile phases consisted of water (A) and acetonitrile (B). Mobile phase A was supplemented with 10 mM ammonium acetate. A gradient of 5–90% B was used for 25 min at a flow rate of 0.3 mL/min. MS was performed using an Agilent 6550 QTOF mass spectrometer equipped with an AJS ESI interface using the following operation parameters: polarity, positive; gas temperature, 250 °C; nebulizer, 35 psig; capillary, (+) 4000 V; and MS and MS/MS range, 100–1500 m/z.

Plant lipids were extracted from 50 mg lyophilised samples of 15-day-old seedlings as described by Matyash et al. (2008 (link)). The Lipidome data were acquired using an Agilent 6530 QTOF for positive ion analysis and Agilent 6550 QTOF Mass spectrometer for negative ion analysis (employing Agilent jet stream thermal focussing technology). Raw data were processed by Agilent's Mass Hunter Qual software to find peaks. Peaks were then imported into Mass Profiler Professional for peak alignments and filtering. To annotate lipids, MSMS files were used to query the Lipid Blast library.

An optimized LC-MS condition established in our lab was employed20 (link). Briefly, chromatographic separation was performed on an Agilent 1290 Infinity UHPLC system (Santa Clara, CA, USA) equipped with a binary solvent delivery system and a standard autosampler. An Agilent Eclipse Plus C18 column (100 × 2.1 mm, 1.8 μm) was employed to separate the SPLs. Detection of SPLs was performed on an Agilent 6550 Q-TOF mass spectrometer (Santa Clara, CA, USA). The Jet Stream electrospray ionization source was operated in positive ion mode. Quantitative analysis was carried out in MRM mode using an Agilent 6460 QQQ mass spectrometer (Santa Clara, CA, USA). The MRM transitions (precursor ion → product ion), fragmentor voltages, and CE values selected for each individual SPLs were given in Table 2.

Sample analysis was performed by UHPLC–qTOF–MS. The instrumentation consisted of an Agilent 1260 infinity II autosampler, Agilent 1290 infinity UHPLC system (1), and an Agilent 6550Q ToF mass spectrometer used in positive ion electrospray mode. Chromatography was performed on a Kinetex 1.7 µm BiPhenyl column (2.1 × 50 mm, Phenomenex, Cheshire, UK). The mobile phase consisted of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The samples were introduced onto the column using 2% mobile phase B at a flow rate of 0.4 mL per minute, followed by a linear gradient to 95% mobile phase B between 0.3 and 2.9 min. The composition was maintained at 95% mobile phase B until 3.55 min, returning to an initial 2% mobile phase B at 3.6 min. Test compounds eluted between 2.3 and 2.5 min. Acceptable mass balance for total compound recovery was defined as 80 to 110%.

Polar metabolite detection was performed on an Agilent 6550 Q‐TOF mass spectrometer operating in negative mode. Metabolites were separated on a SeQuant ZIC‐pHILIC column (5 µM, 150 × 4.6 mm, Millipore) using a binary gradient with a 1200 series HPLC system across a 45‐min method using 20 mM ammonium carbonate (pH 9) and acetonitrile as outlined in Cobbold etal, (2016). Two independent replicates of the metabolite profiling following AMR1 and Lipin depletion were performed using the same ZIC‐pHILIC chromatography on a Thermo Q‐Exactive operating in both positive and negative mode (rapid switching) as described previously (Creek etal, 2016). Lipid extracts were analysed on an Agilent 6550 Q‐TOF using the reverse phase chromatography outlined by Bird etal (2011).
GC‐MS analysis was performed using methods previously described (Saunders etal, 2011). Metabolites were separated using a BD5 capillary column (J&W Scientific, 30 m × 250 µM × 0.25 µM) on a Hewlett Packard 6890 system (5973 EI‐quadrupole MS detector). The oven temperature gradient was 70 °C (1 min); 70°C to 295°C at 12.5°C/min, 295°C to 320°C at 25°C/min; 320°C for 2 min. MS data were acquired using scan mode with a m/z range of 50–550, threshold 150 and scan rate of 2.91 scans/second. GC retention time and mass spectra were compared with authentic standards analysed in the same batch for metabolite identification.