Qp 2010 gc ms instrument

Nine hundred and ninety µL of the volatile extract was added with 10 µL of the internal standard cyclohexanone (1250 µg/mL), and 1 µL of the mixed solution was injected into the QP 2010 GC-MS instrument (Shimadzu, Kyoto, Japan) for analysis using a Rtx-5MS (60 m × 0.32 mm × 0.25 µm) column (Restek Corporation, Bellefonte, PA, USA). Helium was used as the carrier gas at a flow rate of 3 mL/min. The oven temperature was initially programmed at 50 °C for 2 min, then increased from 50 to 200 °C at 3 °C/min, and held at this temperature for 1 min. The temperatures of the ion source and the interface were set at 220 and 250 °C, respectively. The MS was operated in the positive electron ionization mode at 70 eV, and the MS spectra were recorded within an m/z range from 35 to 500 amu.
Most of the compounds were identified by matching their MS spectra and RIs on the Rtx-5MS column to those of standards. The other volatiles that lacked standards were temporarily identified by matching their MS spectra and RIs to those in the mass spectral library (NIST08, NIST08s, FFNSC1.3) and their RI values to those from relevant references. The volatiles with matching standards were quantified according to their respective calibration curves. The concentrations of the other volatiles were estimated using the calibration curve of the internal reference cyclohexanone in scan mode.

Reactions were prepared in sealed vials containing 1 mM SAM, 75 μM GPP, 12.5 μM Sg MT, 10 mM Hepes pH 7.5, 5 mM MgCl₂, 1 mM DTT and 0.5 μM Sg 2-MIBS, 5 μM Sg Enc_2-MIBS, or 5 μM Sg Enc_2-MIBS + 500 μM cAMP. A 100 μL pentane cushion was pipetted on the top of each reaction, and the reactions were incubated for 20 h at room temperature. Samples were centrifuged for 1 min at 14,000 × g, the aqueous layer was transferred to a tube containing 200 μL of pentane, mixed by vortexing, and spun again for 1 min at 14,000 × g, and the pentane layer then pooled with the other pentane layers. 2 to 3 small crystals of disodium sulfate were added to each pentane extract to remove residual water, incubated for 10 min at room temperature, and the pentane layer then transferred to sample vials for GC-MS analysis. The same extraction procedure was used for the 2-MIB standard with a starting concentration of approximately 100 μM. 1 μL of each sample was injected into a Shimadzu QP-2010 GC-MS instrument equipped with a DB-5 column, run in splitless mode with a column flow rate of 0.97 mL/min and a temperature ramp of 60°C to 280°C at 20°C/min. Data analysis was carried out in OpenChrom v 1.5.