Gc 6890

OBD concentrations in abalone and ulva were measured by the following method. Collected samples were lyophilized and powderized. 0.2 g of lyophilized samples were stirred for 24 h in 10 ml of deuterium depleted water at 4°C and lyophilized for three times to remove exchangeable OBD,9 (link) such as deuterium on hydroxyl group or amino group. Then, deuterium concentration was measured by elemental analyzer (vario PORO cube, Elmentar) and mass spectroscopy (IsoPrime 100, Isoprime).
Deuterium concentration in cultivating water was measured by gas chromatography system (GC-6890, Agilent Technologies). Porapak-Q (80/100 mesh, 1/8 in × 6 ft) was used as pre-column and Molecular Sieve 13X (45/60 mesh, 1/8 in × 10 ft) was used as analysis column. Hydrogen gas was used for carrier gas. Deuterium concentration in cultivating water was monitored one time per week. It was checked against and adjusted to designed concentration.

The AITC content in headspace was determined through a septum localized in the lip of a laboratory silo system (Figure 7). The air was recovered using a syringe of 1 mL, and aliquots of 200 µL were injected in a gas chromatograph (GC) with flame ionization detector (FID) (GC 6890, Agilent Technologies Inc., Santa Clara, CA, USA.). The chromatograph was equipped with a 30 × 0.25 mm CP-SIL 88 fused capillary column (Varian, Middelburg, Netherlands). The temperature of the detector arrived at 200 °C with a gradient of temperature that starts at 60 °C. This temperature was maintained for 1 min and increased 8 °C per min up to 100 °C, then maintained for 5 min and finally increased in 15 °C per min up to 200 °C. The gas utilized as the carrier was H₂ at 5 mL/min. The ionization was realized with H₂ at 40 mL/min and purified air at 450 mL/min.

The partially purified active fractions were analyzed by GC–MS. The analyses of the compounds in the active fractions were run on a GC–MS system (Agilent GC: 6890, with a 7683B Autosampler). The fused-silica Rxi-5Sil MS capillary column (30 m 0.25 mm ID, film thickness of 0.25 mm) was directly coupled to an Agilent variant. Oven temperature was programmed (35 °C for 5 min, then 35–300 °C at 10 °C/min) and subsequently, held isothermal for 20 min. The injector port; was 250 °C, the transfer line: 290 °C, splitless. Volume injected: 0.2 ml and the column flow rate was 1 ml/min of 1 mg/ml solution (diluted in chloroform). The peaks of components in gas chromatography were subjected to mass-spectral analysis.
The MS was a LECO Pegasus 4D recording with a EI-source at − 70 eV; the solvent delay was 9 min. Scan time 1.5 s; acquisition rate 10 spectra/second; mass range 50–1000 amu; detector voltage 1800 V, and Ion source temperature: 250 °C. Data were recorded in TIC mode. The software adopted to handle the mass spectra and chromatograms was an Agilent chemstation software. The constituents were identified after comparing with available data in the GC–MS library in the literatures.

COPs were prepared by heating CHO with H₂O₂ at 150°C for 72 h. As evaluated using gas chromatography (GC 6890, Agilent, Wilmington, DE, USA) mass spectrometry, the final extract contained 7α-hydroxycholesterol (8.5%), 7β-hydroxycholesterol (14.8%), 5α- and 6α-epoxycholesterol (18.8%), 5β- and 6β-epoxycholesterol (20.2%), a mixture of 6-ketocholesterol and 25-hydroxycholesterol (6.3%), and 7-ketocholesterol (24.8%).

The extracted lipids were dissolved in hexane to obtain a final concentration of 0.1 g fat/mL hexane and were then used in the following step. We added 1 mL of hexane and 1 mL of a methylating reagent (3N-HCl in methanol) to 100 μL of lipid extracts (of liver) and incubated the samples at 90°C in a water bath for 1 h. After cooling to room temperature, 2 mL of hexane and 5 mL of water were added to the samples, which were mixed thoroughly and incubated at room temperature overnight to allow phase separation. The top hexane layer containing the methylated fatty acids was analyzed for fatty acid composition by using a gas chromatograph (GC 6890, Agilent). Gas chromatography conditions were the same as those used for COPs analysis.

The AITC extraction and quantification from pita bread were carried out as described by Nazareth et al. [35 (link)] with some modifications.
Each pita bread (40 g) was added to hermetic tubes of 150 mL containing 80 mL of methanol. The mixture was extracted for 30 min in a water bath at 40 °C and 10 min in an ultrasonic bath. Then, the extract was centrifuged at 4000× g for 5 min at 20 °C. The supernatant was recovered and filtered through a nylon membrane filter (0.22 μm) and an aliquot of 10 μL was injected in a gas chromatograph.
The residual AITC absorbed by samples was quantified using a gas chromatograph (GC) coupled a flame ionization detector (FID) (GC 6890, Agilent Technologies Inc., Santa Clara, CA, USA), equipped with a fused capillary column (CP-SIL 0.25mm × 30m) (Varian, Middelburg, Netherlands). The inlet temperature was set at 200 °C with 250 °C of detector temperature. H₂ at 5 ml/min was the carrier gas, and the FID gasses were H₂ at 40 mL/min, and purified air at 450 mL/min. The temperature program was a gradient when the initial temperature was 60 °C for 1 min, increased at 8 °C/min up to 100 °C and held for 5 min, then the temperature was raised at 15 °C/min up to 200 °C, the time of analysis was 16.6 min per sample.
Identification and quantification of AITC were carried out comparing the samples areas with points standards curve (1–100 mg/L).