Metabolite extraction and derivatization was conducted as previously described; briefly, a volume of 300μL of the extraction solvent (acetonitrile: water, 1:1 v/v) was added to the cell pellets (2 million cells per pellet), and the supernatants were collected and transferred to gas chromatography (GC) vials for drying using EZ-2 Plus (GeneVac-Ipswich, UK) at 37 ± 1°C (Bataineh MT et al., 2021 (link)). First, the dry samples were dissolved in 25μL of 20 mg/mL methoxyamine hydrochloride in pyridine, followed by the silylation step. Then, for complete derivatization, the samples were incubated at 50°C for 30 min, transferred to 200μL micro-inserts, and analyzed by GC–MS. GC–MS analysis was performed using a QP2010 gas chromatography-mass spectrometer (GC-2010 coupled with a GC–MS QP-2010 Ultra) equipped with an auto-sampler (AOC-20i+s) from Shimadzu (Tokyo, Japan). Chemstation software (Shimadzu). In addition, GC total ion chromatograms (TIC) and fragmentation patterns of the compound were identified using the NIST/EPA/NIH Mass Spectral Library (NIST 14).
Chemstation software
Manufactured by Shimadzu
Sourced in Japan
Chemstation software is a data acquisition and analysis tool developed by Shimadzu for use with their analytical instruments. It provides a platform for collecting, processing, and managing data generated from various Shimadzu chromatography and mass spectrometry systems.
Automatically generated - may contain errors
Lab products found in correlation
Aoc 20i s, by Shimadzu (1 mentions)
Qp2010 ultra, by Shimadzu (1 mentions)
Lc 30ad, by Shimadzu (1 mentions)
Uhplc system, by Shimadzu (1 mentions)
Dgu 20a, by Shimadzu (1 mentions)
Spd 20a, by Shimadzu (1 mentions)
Lc 2030c, by Shimadzu (1 mentions)
Gc 2010 gcms system, by Shimadzu (1 mentions)
Hp 88 fused silica column, by Agilent Technologies (1 mentions)
Qp2010, by Shimadzu (1 mentions)
Gc 2010 gas chromatograph, by Shimadzu (1 mentions)
Aoc 20i autoinjector, by Shimadzu (1 mentions)
Rtx 2330, by Restek (1 mentions)
6 protocols using chemstation software
1
Metabolite Extraction and GC-MS Analysis
2
UHPLC Analysis of Compounds
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A Shimadzu UHPLC system (Japan) was equipped with a Solvent Delivery Pump (Shimadzu LC-30AD), a vacuum degasser (DGU-20A), a Shimadzu UV-VIS spectrophotometric detector (SPD-20A) and ChemStation software (Shimadzu). A Kromasil analytical column was used (5 μm, 150 × 4.6 mm). The mobile phase for UHPLC analysis, consisting of 0.1% acetonitrile-phosphate, was passed under vacuum through a 0.22-μm filter membrane and degassed by exposure to ultrasonic waves before use. UHPLC analysis with ultraviolet detection at 210 nm was performed at a flow rate of 1 mL/min. The column temperature was 35°C. The sample injection volume was 5 μL or 40 μL. The total run time was 55 minutes.
3
HPLC Analysis of Purine Derivatives
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HPLC investigation was performed for PAE, together with reference compounds uracil, hypoxanthine, inosine and gland pyrimidine based on high performance liquid chromatography (HPLC) System (LC-2030C, SHIMADZU, Japan) with an auto-injector sampler programmed at 10 μL, with the use of a C18 column (Agilent five HC-C18 250 × 4.6 mm) at a column temperature of 25°C. All operations, acquisitions and data investigation were regulated with the use of the Chemstation software (SHIMADZU, Japan). The mobile phase was composed by A (methanol: water: acetic acid = 30:69.8:0.2, v/v) and B (methanol: water = 1:1, v/v). Samples were eluted in accordance with the following gradient: 0–10 min 100% A isocratic, 10–20 min 100–70% A, 20–21 min 70–50% A, 21–22 min 50–100% A, 22–25 min 100% A isocratic. The flow rate and the optimized detection wavelength reached 0.6 ml/min and 254 nm, respectively.
4
Quantifying Seed Fatty Acids via GC-MS
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Fatty acids in seeds were measured by gas chromatography as follows. Acid-catalyzed transmethylation reaction on 10 mg of intact seeds was carried out in 5% H2SO4 in methanol (v/v) at 90 °C for 3 h, while 10 μg of heptadecanoic acid was used as an internal control. Fatty acid methyl ester was extracted by hexane and analyzed through a GC-2010 GCMS system (Shimadzu, Japan) equipped with a HP-88 fused silica column (Agilent, USA). The pulsed split mode (250 °C) and helium gas (flow rate of 1 ml/min) were used as the injector condition and carrier gas, respectively, while the pulsed pressure was set at 15 psi for 0.5 min. Scan range was m/z 40–500. The oven program was: 150 °C for 3 min, ramp of 7 °C per min to 240 °C, and keep for 5 min. ChemStation software (Shimadzu, Japan) was used for data acquisition and processing, and compounds were identified by comparison of mass spectrum with authentic standards and the NIST/EPA/NIH mass spectral library v2.0.
5
Fatty Acid Methyl Ester Analysis by GC-MS
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Gas chromatography-mass spectrometry (GC-MS) analysis of FAME was performed with a Shimadzu QP2010 instrument (Shimadzu, Kyoto, Japan). An Rtx-5MS column (30 m × 0.25 mm × 0.25 μm) was used. Oven temperature was raised from 184 to 191 °C by a rate of 2 °C min -1 . Injector and ion source temperature were set at 260 and 200 °C, respectively. Helium was used as a carrier gas with a fl ow rate of 1.30 ml min -1 and a split ratio of 10:1. The identifi cation of components based on NIST05.LIB and NIST05s.LIB with Shimadzu ChemStation software.
6
Plasma Fatty Acid Profiling by GC-FID
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Fast GC analyses were performed on a Shimadzu GC-2010 Gas Chromatograph (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector and a Shimadzu AOC-20i Autoinjector. Separation of FAMEs was carried out on a capillary column (40 m × 0.18 mm I.D. x 0.1 µm film thickness), coated with an RTX-2330 stationary phase of 10% cyanopropyl phenyl-90% byscyanopropyl polysiloxane from Restek (Bellefonte, USA).
Operating conditions were as follows: the split-splitness injector was used in split mode with a split ratio of 1:50. The injection volume of the sample was 1 µL. The injector and detector temperatures were kept at 250 °C and 300 °C, respectively. The temperature program was as follows: initial temperature 110 °C, increased at 52 °C min -1 to 195 °C and held at this temperature for 6 min, and then increased at 25 °C min -1 until 230 °C and held for 6.5 min (total run time: 16.03 min). Hydrogen was used as the carrier gas, at a constant pressure of 26 psi that refers to a linear velocity of 40 cm s -1 at 110 °C. Data acquisition and processing were performed with the Shimadzu-Chemstation software for GC systems. Results were presented as plasma FA concentrations (mg cL -1 ).
Operating conditions were as follows: the split-splitness injector was used in split mode with a split ratio of 1:50. The injection volume of the sample was 1 µL. The injector and detector temperatures were kept at 250 °C and 300 °C, respectively. The temperature program was as follows: initial temperature 110 °C, increased at 52 °C min -1 to 195 °C and held at this temperature for 6 min, and then increased at 25 °C min -1 until 230 °C and held for 6.5 min (total run time: 16.03 min). Hydrogen was used as the carrier gas, at a constant pressure of 26 psi that refers to a linear velocity of 40 cm s -1 at 110 °C. Data acquisition and processing were performed with the Shimadzu-Chemstation software for GC systems. Results were presented as plasma FA concentrations (mg cL -1 ).
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