6890 series gc system

The method of Lu [25 ] was used for gas chromatography/mass spectrometry (GC–MS) analysis with some modification. 20 mg of dry leaves and 1.5 mL extraction solvent (isopropyl alcohol/ACN/water:3/3/2 (v/v/v)) with tridecanoic acid as internal standard were mixed by ultrasound for 1 h. After centrifugation (21 130 g, 10 min), 500 μL of the supernatant was collected and dried, then derivatized at 37 °C for 90 min in 100 μL of 20 mg·mL⁻¹ methoxyamine pyridine (Merck, Darmstadt, Germany) solution and 100 μL of N‐methyl‐N‐(trimethysilyl) trifluoroacetamide (Merck) for 60 min at 60 °C, respectively.
An Agilent 6890 Series GC system combined with an Agilent 5975 series mass selective detector were used. An Agilent DB‐5MS column (0.25 μm, 0.25 mm × 30 m) was chosen. Flow rate was set at 1.2 mL·min⁻¹, 300 °C as the injection temperature, 230 °C as source temperature, and oven temperature programmed at 70 °C for the first 4 min and then increased at 5 °C·min⁻¹ to 310 °C for 15 min. The scan range was set to 33–500 m/z in full scan mode. The data were quantitatively processed with autogcmsdataanal software [26 (link)]; the NIST library (version 2.3) was used for qualitative analysis of metabolites.

Serum (25 µl) collected from ed18 and nd3 broiler birds were spiked with an equal volume of stable isotope-labelled internal standards and were deproteinized with 700 µl of 70% acetonitrile. The samples were centrifuged at 13,500 rpm for 15 min at 4°C, and the supernatant was transferred to a 1 ml v-vial and dried under a stream of nitrogen gas. The metabolites were then converted to their oximes with the addition of 20 µl of 2% methoxamine hydrochloride in pyridine (W/V) and microwaving at 350 W for 90 s. The samples were then derivatized with TBDMS (Tert-butyldimethylsilyl) at 90°C for 1 h. The metabolites were separated on a HP-5MS UI column (30 m × 0.25 mm × 0.25 μm; Agilent, CA, United States) and the ion fragments determined by single ion monitoring (SIM) under electron ionization mode using a GC-MS (5973N, Mass Selective Detector coupled to a 6890 Series GC System, Agilent, CA, United States). Metabolite concentrations were determined in relation to their respective stable isotope-labelled internal standard. For primary hepatocytes, the cells were collected in 1X RIPA and were deproteinized with 700 µl of 70% acetonitrile. The samples were spiked with a known volume of stable isotope-labeled organic acid and amino acid internal standards and sonicated for 10-min to extract the cellular contents. This extract was processed similar to the serum samples for GC-MS analysis.

The measurement of ethylene production was performed according to Xu et al.8 (link). Tomato seedlings were placed in 100 mL gas-tight glass vessels and incubated at room temperature (25 °C) for 2 h. Then, 1 mL sample of gas was removed and analyzed with a flame ionization gas chromatograph (Agilent 6890 Series GC system, Salem, MA, USA) equipped with an activated alumina stainless steel (SS) column. The carrier gas (helium) flow rate was 0.5 mL s⁻¹. The detector and injector were operated at 100 °C, and the oven was at 50 °C.
To determine ACC content and ACS activity, tomato leaves were extracted according to the methods described by Wang et al. 1992 with slight modifications58 (link). Tissues from tomato seedling leaves were extracted in 1 volume of 1.0 M potassium phosphate buffer (pH 8.0) containing 0.5 mM pyridoxal-5-phosphate and 4.0 mM DTE. ACO activity was measured according to Bulens et al.59 (link).

For relative quantification of WE species, yeast WE preparations were dissolved in 200 μl n-hexane and 2 μl of this sample were subjected to GC-FID using a 6890 Series GC System (Agilent) equipped with an Agilent 19091j-413 HP5 5% Phenyl Methyl Siloxane column (30 m x 320 μm x 0.25 μm film thickness; Agilent). Helium was used as the carrier gas (1.5 ml min^-1). The split ratio was 5:1. The temperature gradient was 2 min at 60°C, 60–200°C at 40°C min^-1, 2 min at 200°C, 200–325°C at 3°C min^-1 and 325°C for 16 min. WEs were detected by flame ionization detection.