Agilent 7890a gc

To analyze dark tea samples, a 7890A Agilent GC (Agilent Technologies, CA, USA) system equipped with a 5975C MSD detector (Agilent Technologies), autosampler (7683 B series, Agilent Technologies), and Chemstation software was used. An HP-5MS column (30 m×0.25 mm×0.25 µm film thickness) was used, and the gas carrier (helium at 99.999% of purity) was supplied at a constant rate of 1 mL/min. The injector temperature was 250°C, and the injection mode was splitless. The temperature program began at 50°C for 1 min and increased at a rate of 3°C/min until it reached 210°C (this temperature was held for 3 min), followed by temperature ramping at 15°C/min to a final temperature of 230°C. The mass spectrometer was operated under the electron impact (EI) mode at ionization energy of 70 eV. The aux temperature, MS source temperature, and MS quad temperature were set to 280°C, 230°C, and 150°C, respectively. A mass range of m/z 80–500 was scanned to confirm the retention times of target analytes and data were gathered in full scan mode. The solvent delay time was 2.8 min.

Fatty acid methyl esters were prepared from the extracted lipids using a method based on 14% boron trifluoride–methanol solution [32 (link)]. Nitrogen was used for drying and removing the solvent from fatty acid methyl esters. Obtained samples were analyzed on GC Agilent 7890A system with FID, automated liquid injection module, equipped with capillary column with silica gel (SP-2560, 100 m × 0.25 mm, I.D., 0.20 µm, Supelco Analytical, Bellefonte, PA, United States). Temperature regime during analysis was set as followed: initial temperature was 140 °C with hold of 5 min, heating up to 240 °C was with 2 °C/ min and hold on 240 °C was 5 min. Helium was used as carrier gas (flow rate = 1.26 mL min⁻¹). Fatty acid peaks in samples were identified by comparison with retention times of the standards from Supelco 37 component FAMEs mix and data from internal data library, based on earlier experiments and GC/MS analysis. The results were expressed as a mass of fatty acid or fatty acid group (g) per 100 g of oil.

For in vivo lipolysis assays in mice, serum samples were collected, and free fatty acid levels were then quantified per the manufacturer’s instructions (Biovision Abcam). Serum fatty acids from patients were analyzed by gas chromatography–mass spectrometry performed by the Analytical Facility for Bioactive Molecules platform at the Hospital for Sick Children (Toronto, Ontario, Canada). Briefly, serum samples (20 μL) were spiked with an internal standard mix and acidified with HCl. Nonesterified fatty acids were acidified and double extracted with hexane. The fatty acids were then converted to their pentafluorobenzyl esters using 1% pentafluorobenzyl bromide/diisopropylamine (1:1) and separated by automated gas chromatography (GC Agilent 7890A, Agilent Technologies) on a fused-silica SP2380 capillary column (30 m × 0.25 mm × 0.2 μm film thickness; Supelco Analytical). Fatty acid ions were detected and measured using a MSD Agilent 5975C quadrupole mass detector (Agilent Technologies). Peaks of fatty acid esters were identified by comparisons with individual fatty acid standards (Supelco Analytical).

Reducing sugars were measured using the DNS method [46 (link)], and the standard curves were constructed using 5-fold serial dilutions of a pure chemical sample. The standard curves for glucose, fructose and cellobiose assays were built separately, as these sugars have different reactivities with the DNS reagent [47 (link)]. To prepare samples for ethanol and reducing sugar determination, 1 mL of fermentation broth was centrifuged at 12,800× g for 10 min at 4 °C. The pellet was discarded, and the supernatant was filtered using a 0.22-µm filter (StarTech, Taipei, Taiwan). CO₂ was measured daily, but ethanol and reducing sugars were analyzed using samples that were taken at late stationary phase. These major fermentation end-products were determined by a GC Agilent 7890A (Agilent Technologies, Santa Clara, CA, USA) equipped with a J&W 122-3232: 30 m × 250 µm × 0.25 µm DB-FFAP column, with nitrogen as the carrier gas at a flow rate of 30 mL/min. For ethanol measurement, the front inlet was used, the detector was kept at 225 °C, and the oven was heated from 50 °C to 150 °C (50–100 °C at a ramp rate of 30 °C/min and 100–150 °C at a ramp rate of 20 °C/min). For CO₂ measurement, the back inlet was used, the detector was kept at 225 °C, and the oven was operated isothermally at 50 °C.

A fatty acid profile of the gluten-free cookies was determined following a procedure published earlier [38 (link)]. A chloroform–methanol solution (2:1 ratio of chloroform to methanol) was used to extract the total lipids from samples, and the obtained extracts were dried by vacuum evaporation (40 °C). The solvent was evaporated under a steam of nitrogen and the residue was weighted. The 14% solution of boron (III)-fluoride in methanol was used to convert the extracted lipids into fatty acid methyl esters. The analysis of the samples was performed using GC Agilent 7890A (flame-ionization detector, auto injection module, fused silica capillary column DB WAX 30m, 0.25 mm, 0.50 µm). Helium was used as a carrier gas (purity over 99.9997 vol.%) with a flow rate of 1.26 mL/min. Fatty acids identification was performed by comparing the retention times of samples with those for standards (Supelco 37 Component Fatty Acid Methyl Ester Mix, Sigma-Aldrich, St. Louis, MO, USA). All analyses were performed in triplicates and the results were expressed as g/100 g of sample weight.