Agilent 5975c series

GC-MS analysis of the extracts were carried out in a GC system (Agilent 7890A series, USA) equipped with split/splitless injector and auto-sampler attached to an apolar 5-MS (5% phenyl polymethyl siloxane) capillary column (Agilent 19091S-43; 30 m×0.25 mm i.d. and 0.25-μm film thickness) and fitted to Mass Detector (Agilent 5975C series, USA). The flow rate of the carrier gas, helium (He) was set to be at 1 ml.min⁻¹ in split less mode. The injector temperature was adjusted at 250°C, while the detector temperature was fixed to 280°C. The column temperature was kept at 70°C for 1 min followed by linear programming to raise the temperature from 70° to 200°C (at 8°C min⁻¹ with 2 min hold time), and 200°C to 250°C (at 10°C min⁻¹ with 2 min hold time). The transfer line was heated at 280°C. Total run time was 27.2 min. Mass spectra were acquired in scan mode (70 eV); in the range of 50 to 550 m/z. Twenty microliter each of the extracts (250 mg/ml stock) were further diluted in 2 ml of methanol. One micro liter of this diluted sample was injected for GC-MS analyses.

For the identification of the major components of fraction II, an Agilent Gas Chromatographer 7890A series (Agilent Technologies, Inc., Santa Clara, CA, USA) was used. The injection port and detector temperature was 230 °C. Split (20:1) mode was used for injection. A capillary column HP-5MS 60 m × 0.25 mm inner diameter (i.d.), 0.25 μm thickness (Agilent Technologies, Inc.) was used for the analysis. The oven temperature was programmed as follows: initial temperature of 40 °C for 5 min; then, temperature was risen at a rate of 5 °C/min until 230 °C and maintained for 15 min. As a carrying gas, helium was used at a constant flow of 1 mL/min. The relative abundance of each compound was determined from its area under the curve. Mass spectrometry was achieved using an electron impact mass spectrometer Agilent 5975C series with a quadrupole analyzer. The temperatures of the ion source and the quadrupole were 230 and 250 °C, respectively. The transfer line was set at 280 °C. A complete screening was used in a rank 33 to 300 amu, with a scanning rate of 5.2 s⁻¹ and an ionization voltage of 70 eV. The MSD Chemstation E.0100237 software was employed for data analysis. Compound identification was performed by comparing spectral data with data from the NIST/EPA/NIH Mass Spectra Library, version 1.7, USA.