7890 gc system

Metabolomics analyses were performed using gas chromatography–mass spectrometry (GC–MS) as previously described^{75 (link)}. Briefly, serum samples were collected from animals at the time of death. A total of 25 µl of serum was mixed with 200 µl of 100% ice-cold methanol. Norvaline and D27-myristic acid were used as spike-in controls. The samples were frozen for at least 24 h at –80 °C. The mixture was centrifuged (20,000g, 4 °C, 5 min), and the supernatant was kept for analysis. For measurement, the supernatant was dried in a speed-vac (room temperature), and samples were resuspended in 10 µl of pyridine with 10 mg ml⁻¹ methoxyamine and incubated for 1 h at 30 °C. Samples were centrifuged (20,000g, 3 min), and the supernatant (7.5 µl) was transferred to GC–MS tubes. Subsequently, metabolites were derivatized by the addition of 15 µl of N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamid with 1% tert-butyldimethylchlorosilane (Sigma-Aldrich, 375934) and incubation for 60 min at 80 °C. Metabolites were measured using a DB5-MS GC column in a 7890 GC system (Agilent Technologies) combined with a 5977 MS system (Agilent Technologies).

The fat, protein, ash, and moisture content of the raw materials were measured using official AOAC methods (AOAC 2000). Fatty acids (FAs) of the SBO were determined according to the method of AOAC 969.33 (2000) by Gas chromatography (7890 GC system, Agilent Technologies Inc., Santa Clara, CA, USA). The initial amount of starch in the samples was evaluated using an analytical kit for total starch.

GC-MS analysis was conducted on an Agilent 7890 GC system coupled with an Agilent 5975C mass analyzer (Agilent, Palo Alto, USA). Chromatographic separation was carried on an Agilent DB-5MS capillary column (30 m × 0.25 mm ID × 0.25 μm film thickness). One microliter of the derivatized sample or reference standard was injected in the splitless mode. Helium was used as carrier gas with flow rate of 1.5 mL/min. The temperatures of inlet, transfer line, and ion source were maintained at 250, 300, and 230°C, respectively. The GC temperature programming was set to 4 min isothermal heating at 60°C, followed by the first ramp at 6°C/min to 150°C and holding for 8 min, second ramp at 8°C/min to 280°C and holding for 10 min, and third ramp at 12°C/min to 320°C. Mass was in the electron impact mode at 70 eV and in the full-scan monitoring mode from m/z 35 to 750. The 1701EA station software (Agilent, Palo Alto, USA) was used to acquire chromatogram and detect mass spectral peaks.

Plasma n-3 and n-6 PUFA were quantified from baseline specimens prior to CAD events, in a targeted mode using gas chromatography–mass spectrometry (GC-MS)/MS on an Agilent 7890 GC system (Shanghai, China) equipped with a G7000B QQQ triple quadrupole mass detector and an auto sample injector. Both free and esterified (triglycerides, phospholipids, cholesterol esters) FA fractions were measured in total. Samples were analyzed in 76 batches, with cases and matched controls included in the same batch. Pooled human plasma was used for quality control (QC). The experimental details and the coefficients of variation of the measured FAs were published elsewhere [20 (link)].