Hp 6890

Constituents of tea tree extract were analyzed by gas chromatography–mass spectrometry (GC-MS) (Hewlett-Packard Co., Palo Alto, CA, USA) using HP 6890 (gas chromatograph) and HP 5973 (mass spectrometer) models. About 1 μL of tea tree extract sonicated with hexane was used, and detailed information of GC–MS analysis is summarized in Table 2. α-Terpineol (96% purity, Alfa Aesar, Ward Hill, MA, USA), 1,8-cineole (99% purity, Alfa Aesar, Ward Hill, MA, USA), terpinen-4-ol (≥95% purity, Sigma Aldrich, Burlington, MA, USA), α–terpinene (≥95% purity, Sigma Aldrich, Burlington, MA, USA), and γ–terpinene (97% purity, Sigma Aldrich, Burlington, MA, USA) were analyzed as reference materials to determine the constituents. These organic materials have been known for the main phytochemical constituents of tea tree extract [20 (link)].

Lipids from blood plasma samples and biceps femoris muscle were extracted and methylated using the procedure described by [22 (link)]. Fat extracts were methylated in the presence of sulfuric acid and analyzed by gas chromatography. Previously fatty acid methyl ester (FAME) samples were identified by gas chromatography, as described elsewhere [23 (link)]. GC-MS was performed using an HP-6890 (Hewlett Packard, Avondale, PA, USA) gas chromatograph, equipped with a flame ionization detector and capillary column (HP-Innowax, 30 m by 0.32 mm ID and 0.25 μm polyethylene glycol-film thickness). A temperature program of 170 °C to 245 °C was used. The injector and detector were maintained at 250 °C. The carrier gas (helium) flow rate was 2 mL/min. For the identification of each fatty acid, pure standards were used (Sigma). The concentration of individual fatty acids was calculated as a % of total fatty acids. The results were expressed as grams per 100 g of detected FAMEs.

Fasting blood samples were taken between the 26th and the 28th week of gestation. Plasma was prepared by centrifugation and was stored at −80°C until analysis. Total lipid extraction was carried out with chloroform/methanol (2:1 v/v) and PC, which contributes about 75% of plasma phospholipids, was isolated by solid-phase extraction on aminopropylsilica cartridges and eluted with chloroform/methanol (3:2 v/v). Fatty acid methyl esters were generated by reaction of purified PC with 2% sulfuric acid (v/v) at 50°C for 2 hours, extracted into hexane and separated by gas chromatography. A BPX-70 column (30 m × 220 μm; film thickness 0.25 μm) fitted to a Hewlett-Packard HP6890 gas chromatograph was used for separation with helium as the running gas and detection of fatty acid methyl esters by flame ionization before quantification using the ChemStation software in absolute concentration (μg/mL plasma). Plasma PC fatty acids were expressed as percentages of total plasma PC fatty acids, and the ratio of total n-3 to n-6 PUFAs were calculated accordingly.

The chemical components of E. globulus EO were determined using gas chromatography coupled to mass spectrometry (GC/MS) analysis conditions as described by Mekkaoui et al. [27 (link)]. Briefly, a Hewlett-Packard (HP6890) GC instrument coupled with an HP5973 MS and equipped with a 5% phenylmethyl silicone HP−5 MS capillary column (30 m × 0.25 mm × film thickness 0.25 μm) was used in GC analysis. The column temperature was increased from 50 °C for 5 min to 200 °C with a 4 °C/min rate. Helium with a 1.5 mL/min flow rate and split mode (flow: 112 mL/min, ratio: 1/74.7) was the carrier gas used. The hold time was 48 min, while the temperature of the injector and detector was 250 °C.
The machine was led by the computer system ″HP ChemStation″, managing the functioning of the machine and allowing us to follow the evolution of chromatographic analyses. Diluted samples (1/20 in methanol) of 1 μL were injected manually. In addition, 70 eV ionization voltage, 230 °C ion source temperature, and a 35–450 (m/z) scanning range were the MS operating conditions. Finally, the qualitative quantification of the different compounds was based on the percent area of each peak of the sample compounds and was confirmed by reference to their MS identities (Library of NIST/EPA/NIH MASS SPECTRAL LIBRARY Version 2.0, build 1 July 2002).

The product’s fatty acid profile was calculated by isolating lipids from samples by chloroform/methanol extraction, as described by the Folch method. Thin-layer chromatography was utilized to examine the extracted lipids for purity. Fatty acid composition was measured using an Agilent Technologies, USA HP-Innowax 30 m × 0.25 mm × 0.25 m column on a Hewlett Packard HP 6890 gas chromatograph with a flame ionization detector. The analysis was performed in triplicate.

The one-step procedure [44 (link)] was used for the extraction and methylation of the diet fatty acids (FA). Gas chromatography (Hewlett Packard HP-6890, Palo Alto, CA, USA) was used to identify fatty acid methyl esters by a flame ionization detector and a capillary column (HP-Innowax, 30 m × 0.32 mm i.d., and 0.25 µm polyethylene glycol-film thickness; [45 (link)]).
The lipids from the brain, intramuscular fat (IMF) at the LD muscle, and liver fat were extracted [46 (link)]. Fat content was expressed as a percentage (%) of dry matter (DM). Afterward, total lipids at IMF and liver fat were separated into the neutral lipid (NL; in fat storage such as triglycerides) and polar lipid fractions (PL; in cell membranes such as phospholipids; [47 (link)]). Subcutaneous fat was individually analyzed in outer and inner layers. Extracts were methylated [48 (link)] and analyzed using protocols developed at our laboratory [45 (link)]. The individual FA percentages for saturated, monounsaturated, and polyunsaturated FA (SFA, MUFA, and PUFA) were calculated. Total n-3, total n-6 FA, the Σn-6/Σn-3 ratio, and the unsaturated index (UI) were also calculated [49 (link)]. The activity of stearoyl-CoA desaturase enzyme 1 (SCD1) was estimated as C18:1/C18:0 and MUFA/SFA ratios (desaturation indexes; [50 (link)]).