Agilent 5973 mass selective detector

Gas samples were taken by using a syringe to draw 120 mL of the headspace in the packages through an adsorbent tube packed with Tenax GR for trapping the volatile compounds. The adsorbent tubes were desorbed at 280 °C for 7 min in a Markes Thermal Desorber and transferred to an Agilent 6890 GC with an Agilent 5973 Mass Selective Detector (EI, 70 eV). The volatiles were separated on a DB-WAXetr column (30 m, 0.25 mm i.d., 0.5 µm film) with a temperature program starting at 30 °C for 10 min, increasing 1/min to 40 °C, 3/min to 70 °C, and 6.5/min to 230 °C, hold time 5 min. The peaks were integrated, and compounds tentatively identified with HP Chemstation software and NIST 2011 Mass Spectral Library. The volatile compounds are expressed in arbitrary units of the deconvoluted component of the peak area. The result shown are an average of three replicates.

GC–MS analysis was performed using an Agilent 6890N Network GC System (Agilent Technologies, Santa Clara, California) coupled to an Agilent 5973 Mass Selective Detector (Agilent Technologies, Santa Clara, California). Chromatographic separation was performed by an HP 5 MS GC Column, 30 m x 0.25 mm x 0.25 μm (Agilent Technologies, Santa Clara, California). Hereby an initial step of 60°C for 1 min was succeeded by a temperature ramp of 30°C/min up to a temperature of 225°C and a subsequent temperature ramp of 75°C/min up to a temperature of 300°C, which was held for 5 min. The mass spectrometer was operated in selected ion monitoring mode with m/z ratios for propofol detection of 117.1, 163.2, and 178.1 and included a solvent delay of 3 min and a dwell time of 10 ms. Laminar flow in in vitro microdialysis experiments for permeability testing was obtained using a Harvard Apparatus standard infusion syringe pump (Harvard Apparatus, South Natick, Massachusetts).

Snap-frozen tissues collected from different tumor-bearing mice, or cell samples were homogenized in ice-cold methanol. Metabolite extraction for LC-MS/MS analysis was prepared as previously described [13 (link)]. Peaks were normalized against the total ion count and tissue weight. For ¹³C enrichments, homogenates were subjected to three rapid freeze-thaw cycles by transferring them from liquid nitrogen to a 37 °C water bath. Samples were centrifuged at 13,000g at 4 °C for 15 min, and the supernatant transferred to a screw-topped glass tube with 50 nM of sodium-2-oxobutyrate then completely evaporated at 42 °C under blown air. Evaporated samples were re-suspended in 30 μl pyridine containing methoxyamine (10 mg/ml). After 10 min at 70 °C, 70 μl of MTBSTFA reagent was added and heated at 70 °C for 1 h. GC-MS was performed using an Agilent 6890N Gas Chromatograph coupled to an Agilent 5973 Mass Selective Detector (Agilent Technologies, Santa Clara, CA). One microliter of each standard or sample was injected and analyzed in scan mode.

The FAME was subjected to GC-MS detection performed with Agilent 6890N Gas Chromatograph connected to Agilent 5973 Mass Selective Detector at 70 eV (m/z 50–550; source at 230°C and quadruple at 150°C) in the electron impact mode with a HP-5 ms capillary column (30 m × 0.25 mm i.d. × 0.25 μm film thickness). The oven temperature was programmed for 2 min at 160°C and raised to 300°C at 5°C/min and maintained for 20 min at 300°C. The carrier gas, helium, was used at a flow rate of 1.0 mL/min. The inlet temp was maintained at 300°C, and the split ratio was 50 : 1. Structural assignments were based on interpretation of mass spectrometric fragmentation and confirmed by comparison of retention times as well as fragmentation patterns of authentic compounds. GC analysis was performed on a HP 6850 Series gas chromatograph equipped with a FID detector and DB-225 capillary column (30 m × 0.25 mm I'd. × 0.25 μm film thicknesses). The injector and detector temperatures were maintained at 300 and 325°C, respectively. The oven temperature was programmed for 2 min at 160°C and raised to 300°C at 5°C/min and maintained for 20 min at 300°C. The carrier gas, nitrogen, was used at a flow rate of 1.5 mL/min. The injection volume was 1 μL, with a split ratio of 50 : 1. The identification of individual fatty acids was done on the basis of retention time.

After 8 days of fermentation, mycelia in the fermentation broth were collected. The fatty acid in cell membrane of the mycelia was extracted, purified and methylated according to the method described by Wang et al. (2013 (link)). After that, the sample dissolved in the n-hexane was collected for GC–MS analysis, using an Agilent 6890 GC (Agilent, Santa Clara, CA, USA) coupled to an Agilent 5973 mass selective detector (MSD) (Agilent, Santa Clara, CA, USA), equipped with a HP-5MS column (5% Phenyl Methyl Silox, 30 m–0.25 mm id 0.25 μm film thickness, Agilent, Santa Clara, CA, USA). The front injection was 250 °C with a split ratio of 70:1. Helium gas (purity of 99.9999%, Foshan, China) was used as the carrier gas at a flow rate of 50 mL/min. The oven temperature program was as follows: 80 °C for 2 min, then raised to 150 °C at a rate of 10 °C/min, and then further to 230 °C at a rate of 3 °C/min, keeping at 230 °C for 5 min. The electron impact energy was 70 eV, and the ion source temperature was set at 230 °C.

Cell samples were homogenized in ice-cold methanol. Metabolite extraction for LC-MS/MS analysis was prepared as previously described^{79 (link)}. GC-MS was performed using an Agilent 6890N Gas Chromatograph coupled to an Agilent 5973 Mass Selective Detector (Agilent Technologies, Santa Clara, CA).

FAXs extracted from NNMB and NMB were analyzed for monosaccharide composition. Individual neutral sugars in the extracted FAXs were determined after hydrolysis (1 M H₂SO₄, 100 °C, 90 min) and converted to alditol acetates as followed by Pettolino et al. [9 (link)]. Gas chromatography–mass spectrometry (GC-MS) (Agilent 6890) coupled with Agilent 5973 Mass Selective Detector was used to determine arabinose, xylose, galactose and glucose. For this purpose, a 30 m long DB-5 fused silica capillary column and film thickness of 1.0 µm and a 0.25 mm inside diameter with methyl polysiloxane polymer phase were used. The detector and injection port temperatures were 300 °C and 280 °C, respectively. Nitrogen was used as the carrier gas with a flow rate of approximately 1.5 mL/min. The GC-MS was operated in the electron impact mode with an ionization energy of 70 eV. A 2.0 µL splitless sample was injected to quantitate individual sugars. Monosaccharide experiments were repeated three times for accuracy.