5975b mass spectrometer

Analysis of the compounds in the volatile oil was performed on Agilent 6890N gas chromatograph (GC) combined with a 5975B mass spectrometer (MS) equipped with a HP-5MS column (30 m × 0.25 mm × 0.25 μm). High-purity helium was used as the carrier gas at a constant flow rate of 1.0 mL/min, and the inlet temperature was 250°C. The temperature-increasing procedure was set as follows: started at 40°C and maintained for 3 min, and then raised to 160°C at a rate of 3°C/min and kept it for 2 min; it was then raised to 220°C at a rate of 8°C/min and held for 3 min. The detection of the volatile oil was in the full-scan mode with the ion source temperature of 230°C and quadrupole temperature of 180°C. The collected mass spectrogram was searched using the NIST spectral database to identify the components in the volatile oil, and the relative contents of each component were analyzed by the area normalization method.

Volatiles were sampled by SPME with a 2 cm × 50/30-micron DVB/Carboxen/PDMS Stable Flex fiber (Sigma, Milano, Italy). Extraction and desorption of the volatiles were performed automatically by a CombiPAL autosampler (CTC Analytics, Zwingen, Switzerland) as described (Zorrilla-Fontanesi et al., 2012 (link)). Chromatography was performed on a DB-5 ms (30 m × 0.25 mm × 1 mm) column (Sigma, Milano, Italy) with Helium at a constant flow of 1.2 mL/min, accordingly to Zorrilla-Fontanesi et al. (2012 (link)). Mass spectra were recorded in scan mode in the 35 to 220 mass-to-charge ratio range by a 5975B mass spectrometer (Agilent Technologies, Cernusco sul Naviglio, Italy) (ionization energy 70 eV; scanning speed 7 scans/s). The Enhanced ChemStation software (Agilent Technologies, Cernusco sul Naviglio, Italy) was used for recording and processing of chromatograms and spectra. Three technical replicas were conducted for each sample.

50 mg of liver sample was added to 150 μl of physiological saline for homogenate. Then 500 μl methanol were added, followed by 1 min of vortex mixing and 10 min of standing at -20 ℃ for protein precipitation. After centrifugation at 12000 g for 10 min at 4 ℃, 300 μl of the supernatant was transferred to an autosampler vial and blown to dryness with nitrogen. The residue was dissolved in 80 μl of methoxyamine (15 mg/ml) and the methoximation reaction was carried out for 90 min of shaking at 30 ℃, then 50 μl of BSTFA containing 1% TMCS was added for another 1 h of thimethylsilylation at 70℃. At last, a 1 μl of aliquot of the solution was injected into an Agilent 6890 GC system coupled with 5975B mass spectrometer (Agilent technologies, USA) for analysis.
Chromatographic separation was carried out on a capillary column (Agilent J&W DB-5ms Ultra Inert, 30 m * 0.25 mm * 0.25 μm) using programmed temperature showed in Table 1. Parameters in mass spectrometer were as follows: scan range, m/z 30–550; temperature of injection, interface and source, 280 ℃, 260 ℃ and 230 ℃.

An Agilent Technologies 6890 N (Agilent Technologies Deutschland GmbH, Böblingen, Germany) gas chromatograph was coupled to an Agilent Technologies 5975B mass spectrometer. Separation of the analytes was performed on a ZB-1MS capillary column (30 m × 0.25 mm; ft 0.25 µm; Phenomenex, Aschaffenburg, Germany). The following temperature program was used: 100 °C (3 min)–6 °C/min–310 °C (3 min). The following MS settings were applied: ionization voltage of 70 eV, ion source temperature of 230 °C, and interface temperature of 290 °C. A set of co-injected hydrocarbons (evenly numbered C14–C32) was used to calculate the retention indices by linear extrapolation, as described by Frölich et al. [74 (link)]. The PA compounds were identified by retention indices and an in-house mass spectra library of authentic reference compounds. PA patterns of the individual plant samples were compared using the Agilent ChemStation software (Version D.03.00.611). The respective ratio (relative abundance, RA) of each PA was calculated based on its TIC-area signal in relation to the sum of all TIC-area signals of all PAs of the sample.

For quantitative analysis, the samples for terpenoid identification using gas chromatography-mass spectrometry (GC/MS) were prepared according to Karanikas’ method [20 ].
Subsequently, terpenoids of masson pine were detected by GC/MS with an Agilent 6890N gas chromatography coupled with a HP-5MS column (ID: 0.25 mm, length: 30 m, film thickness: 0.25 μm), and an Agilent 5975B mass spectrometer. For GC, the program of oven temperature was 60°C for 2 min, increasing 2°C/min to 80°C, 80°C for 5 min, subsequent increase rate of 4°C/min until 280°C and 280°C for 5 min. Helium was used as the carrier gas at a flow rate of 1 mL/min, injector temperature was set up at 260°C, injection volume was 1 μL with a split ratio of 1:50. Electron ionization mass spectrometry analysis was carried out with 70 eV electron energy, 230°C ion source and 280°C connection part temperature. Terpenoids were identified by matching fragmentation of mass spectra with NIST08. In addition, experimental retention indices were also used to match with reference compounds. The concentration of each terpenoid was expressed as the mass of a compound in oleoresin per gram. SPSS 18.0 statistical package was used to perform the statistical analysis. The significant difference between high and low oleoresin-yielding clones was determined by non-parametric Mann-Whitney test.

All GC-MS analyses were performed by a 5975B mass spectrometer (Agilent technologies, USA) coupled to an Agilent 6890 (Agilent technologies, USA) GC instrument using a capillary column (Agilent J&W DB-5ms Ultra Inert 30 m × 0.25 mm, film thickness 0.25 μm). Helium carrier gas was used at a constant flow rate of 1.0 mL/min. One microliter of each derivatized sample was injected into the GC-MS instrument using the splitless injection mode. A column temperature program, as listed in Table S2, was optimized to acquire good separation. The injection port, interface and source temperature was set at 270°, 260°, and 220°C, respectively. The measurements were made with electron impact ionization (70 eV) in the full scan mode (m/z 30–550). The solvent post time was set to 5 min. The GC-MS operating conditions were the same as those in a previous experiment (Sun et al., 2012 (link)) except the column temperature program. Metabolites were then identified by searching in the NIST 2011 database and verified by standards.