Rxi 17sil ms

Metabolite profiling of the endophyte extracts was carried out on a GC-TOF-MS system (LECO Corporation St. Joseph, MI, United States) using the following conditions: primary column and a Rxi-5Sil MS (30 m, 250 μm i.d., 0.25 μm d_f) (Restek, Pennsylvania, United States) and a Rxi-17Sil MS (2 m, 250 μm i.d., 0.25 μm d_f) (Restek, Bellefonte, PA, United States) secondary column. In brief, samples were first prepared by adding 1 ml HPLC grade methanol (Sigma-Aldrich, Aston Manor, South Africa) to the extracts, 1 μl of each sample was injected, and Helium was used as a carrier gas with a flow rate of 1 ml/min. The oven temperature was maintained at 60°C for 1 min and then programmed at 10°C/min increment to 330°C, then 5°C/min to 280°C. The inlet temperature was 250°C. Mass spectra (MS) were optimized at an electron energy of −70 eV with an ion source at 250°C. The mass fragments used were from 40–660 m/z with an acquisition rate of 10 spectra/second. The interpretation of GC-MS mass-spectra was analyzed using the ChromaTOF software (LECO Corporation, St. Joseph, MI, United States). The functional groups and biological activities of the compounds were analyzed using the NCBI PubChem and PASS online databases available at https://pubchem.ncbi.nlm.nih.gov and http://www.way2drug.com/passonline, respectively.

All GC × GC-MS applications were carried out on a 7890B chromatography system (Agilent Technologies, Santa Clara, CA, USA) and a time-of-flight mass spectrometer Pegasus BT 4D (LECO Corporation, Benton Harbor, MI, USA) equipped with an L-PAL3 autosampler (CTC Analytics AG, Zwingen, Switzerland).
Each sample (1 µL) was injected through the glass liner (Restek, Bellefonte, PA, USA) under split mode (50:1). Helium (6.0 grade) was used as a carrier gas, and its constant flow of 1 mL/min was maintained throughout the run. The oven was initially heated to 60 °C, the equilibration time was 1 min, and the temperature was ramped up at the rate of 10 °C/min to the final temperature of 280 °C, with a hold time of 12 min. The first-dimension column was 30 m-long Restek Rxi-5Sil MS (Restek, Bellefonte, PA, USA, catalog #13623), and the second-dimension column was 3 m-long Restek Rxi-17Sil MS (Restek, Bellefonte, PA, USA, catalog #15123).
The transfer line of the time of MS was set at 280 °C, with a solvent delay of 350 s. The ion source temperature was 250 °C. Spectra were collected from 35–700 m/z at 70 eV electron ionization energy. The scan rate was 200 spectra per second. Data were acquired by ChromaTOF software (v. 5.51, LECO Corporation, Benton Harbor, MI, USA).

The contents of cholesterol and oxysterols were determined according to the slightly improved method of Czauderna et al. [32 (link)]. The improvements consisted of silylation with a BSTFA (N,O-Bis(trimethylsilyl)trifluoroacetamide, 99%; Sigma Aldrich, St. Louis, MO, USA) procedure. Derivatized analytes were separated on a GC-TOFMS Pegasus^® BT (LECO Corporation, St. Joseph, MI, USA) chromatograph equipped with a capillary column (30 m × 0.25 mm × 0.25 μm film thickness, Rxi^®-17SilMS, Restek, Bellefonte, PA, USA). Identification was made on the basis of mass spectra and by a comparison of retention times of analytes with the following standards: cholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, cholesterol 5α,6α-epoxide, 7-ketocholesterol; Sigma, USA). For recoveries, 5α-cholestane (Sigma, St. Louis, MO, USA) as an IS was used.