192
9.2.3 Instrumentation
9.2.3.1 GC-MS
The samples were run on a 7890A GC coupled to a 5975C MSD (Agilent); the column used was a VF 5 ms 30 m × 250 μm × 0.25 μm with a 10 m guard column
(Agilent J&W). The carrier gas was helium and the column flow was 1 mLmin
−1
.
The temperature gradient of the oven was set initially at 70 °C for 1 min and then
increased by 7 °C per minute to 325 °C. The inlet, thermal auxiliary, MS source and
MS quadrupole temperatures were set to 250 °C, 280 °C, 230 °C and 150 °C,
respectively. The scan range was m/z 50–600. Metabolites were identified in ADMIS
version 2.72 (NIST) using the Metabolomics Australia in-house library and NIST
14 library version 2.2 for mass spectral comparison.
9.2.3.2 LC-MS
Instrument and LC-MS setup were as follows: LC-MS system (Agilent 6520 QTOF,
Santa Clara, CA, USA) with a dual sprayer ESI source and attached to Agilent 1200
series HPLC system (Santa Clara, CA, USA) comprised of a vacuum degasser,
binary pump, with a thermostated autosampler and column oven. The MS was operated in positive mode using the following conditions: nebulizer pressure 30 psi, gas
flow rate 10 L/min, gas temperature 300 °C, capillary voltage 4000 V, fragmentor
150 and skimmer 65 V. The instrument was operated in the extended dynamic range
mode with data collected in m/z range 70–1700. Chromatography was carried out
using Zorbax Eclipse XDB-C18 column (2.1 × 100 mm, 1.8 μm column, Agilent)),
maintained at 40 °C (± 1 °C) at a flow rate of 400 μL min
−1
with a 20-min run time.
A gradient LC method was used with mobile phases comprised of (A) 0.1% formic
acid in deionized water and (B) 0.1% formic acid in acetonitrile. Gradient: A 5-min
linear gradient from 5% solvent (B) to 30% solvent (B), followed by a 5-min linear
gradient to 30% solvent (B) to 100% solvent (B), and then a 5-min hold at 100%
solvent (B) and a 4-min re-equilibration at 5% solvent (B). Additionally, targeted
MS/MS analysis was performed using Agilent 6510 Q TOF (Santa Clara, CA, USA)
following the chromatographic conditions as detailed above. The MS parameters
used were as follows: m/z 100–950 (MS/MS); capillary voltage, 3500 V; nebulizer
pressure, 30 psig; gas flow, 5 L/min; gas temperature, 325 °C; fragmentor voltage,
160 V; collision energy, 15 and 25 eV; and skimmer voltage, 65 V.
9.2.4 Chromatographic Peak Identification
Molecular feature extraction was conducted using Agilent MassHunter Profinder
(version B.06.00) and MassHunter Qualitative analysis (version B.07.00). Putative
molecular feature identification was done by accurate mass (<10 ppm) and retention
time (<0.3 min) matching using an in-house library maintained at Metabolomics
U. Kuzhiumparambil et al.
9.2.3 Instrumentation
9.2.3.1 GC-MS
The samples were run on a 7890A GC coupled to a 5975C MSD (Agilent); the column used was a VF 5 ms 30 m × 250 μm × 0.25 μm with a 10 m guard column
(Agilent J&W). The carrier gas was helium and the column flow was 1 mLmin
−1
.
The temperature gradient of the oven was set initially at 70 °C for 1 min and then
increased by 7 °C per minute to 325 °C. The inlet, thermal auxiliary, MS source and
MS quadrupole temperatures were set to 250 °C, 280 °C, 230 °C and 150 °C,
respectively. The scan range was m/z 50–600. Metabolites were identified in ADMIS
version 2.72 (NIST) using the Metabolomics Australia in-house library and NIST
14 library version 2.2 for mass spectral comparison.
9.2.3.2 LC-MS
Instrument and LC-MS setup were as follows: LC-MS system (Agilent 6520 QTOF,
Santa Clara, CA, USA) with a dual sprayer ESI source and attached to Agilent 1200
series HPLC system (Santa Clara, CA, USA) comprised of a vacuum degasser,
binary pump, with a thermostated autosampler and column oven. The MS was operated in positive mode using the following conditions: nebulizer pressure 30 psi, gas
flow rate 10 L/min, gas temperature 300 °C, capillary voltage 4000 V, fragmentor
150 and skimmer 65 V. The instrument was operated in the extended dynamic range
mode with data collected in m/z range 70–1700. Chromatography was carried out
using Zorbax Eclipse XDB-C18 column (2.1 × 100 mm, 1.8 μm column, Agilent)),
maintained at 40 °C (± 1 °C) at a flow rate of 400 μL min
−1
with a 20-min run time.
A gradient LC method was used with mobile phases comprised of (A) 0.1% formic
acid in deionized water and (B) 0.1% formic acid in acetonitrile. Gradient: A 5-min
linear gradient from 5% solvent (B) to 30% solvent (B), followed by a 5-min linear
gradient to 30% solvent (B) to 100% solvent (B), and then a 5-min hold at 100%
solvent (B) and a 4-min re-equilibration at 5% solvent (B). Additionally, targeted
MS/MS analysis was performed using Agilent 6510 Q TOF (Santa Clara, CA, USA)
following the chromatographic conditions as detailed above. The MS parameters
used were as follows: m/z 100–950 (MS/MS); capillary voltage, 3500 V; nebulizer
pressure, 30 psig; gas flow, 5 L/min; gas temperature, 325 °C; fragmentor voltage,
160 V; collision energy, 15 and 25 eV; and skimmer voltage, 65 V.
9.2.4 Chromatographic Peak Identification
Molecular feature extraction was conducted using Agilent MassHunter Profinder
(version B.06.00) and MassHunter Qualitative analysis (version B.07.00). Putative
molecular feature identification was done by accurate mass (<10 ppm) and retention
time (<0.3 min) matching using an in-house library maintained at Metabolomics
U. Kuzhiumparambil et al.