The main components of the EOs were analyzed by gas chromatography/mass spectrometry with an Agilent 5977A Series GC/MSD System as previously described28 (link).
5977a series gc msd system
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The 5977A Series GC/MSD System is a gas chromatography-mass spectrometry (GC/MS) instrument designed for analytical applications. It combines a gas chromatograph and a mass spectrometer to separate, identify, and quantify chemical compounds in complex mixtures. The system provides high-performance separation, detection, and data analysis capabilities.
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8 protocols using 5977a series gc msd system
1
Gas Chromatography-Mass Spectrometry Analysis
2
Essential and Fixed Oil Extraction
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Essential oil and fixed oil were extracted through Clevenger apparatus and
Soxhlet extractor, respectively.13 (link) While qualitative and
quantitative analysis were done by using Gas chromatography and Mass
spectroscopy (5977A Series GC/MSD System, Agilent Technologies, Inc.,
USA).14 (link)
Soxhlet extractor, respectively.13 (link) While qualitative and
quantitative analysis were done by using Gas chromatography and Mass
spectroscopy (5977A Series GC/MSD System, Agilent Technologies, Inc.,
USA).14 (link)
3
Yeast VLCFA Analysis by GC-MS
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Samples were analyzed using the Agilent 5977A series GC/MSD system fitted with a 30 m × 320 μM × 1 μM HP-5 column. The initial injection temperature of 150 °C was held for 1 min, ramped to 280 °C at a rate of 4 °C/min, then held at 280 °C for 7 min with a helium gas flow rate of 1.2 ml/min. Compound peaks from the resulting chromatograms were identified using mass spectra and retention time comparison with authentic standards (Fig. S5 ). Peak areas were normalized to the weight of lyophilized yeast, internal and external standards (methyl heptadecanoate for saturated VLCFAs and methyl octadecenoate for unsaturated VLCFAs) to obtain compound abundance.
4
Metabolomic Analysis of Mouse and Zebrafish Hearts
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Frozen zebrafish heart tissue (20–25 mg) specimens were mixed with a 1.2 mL mixture of cold methanol and chloroform (1:2 v/v; 4°C) and homogenized using tissue teAror homogenizer and sonicated for 20 seconds (Marney et al., 2013 (link)). A further 800 μL cold chloroform/distilled water solution (1:1 v/v) was added, the sample was then vortexed and the set aside for 30 minutes on ice to separate the solvent layers. Next, after centrifugation (2000 rpm), the aqueous (top) layer was separated and filtered using 1.5 mL 0.2 μm syringe filter and freeze dried.
To prepare the samples for GC/MS analysis, 30 uL of 20 mg/mL methoxyamine hydrochloride dissolved in pyridine was added to the dried aqueous metabolites, and samples were incubated at 37°C for 90 minutes. MTBSTFA (70 uL) was subsequently added and incubated at 37°C for 30 minutes. Samples were run on an Agilent 5977A Series GC/MSD system.
N = 2 for adult mouse heart, N = 3 for P7 mouse heart and N = 4 for P0.5 mouse heart.
N = 2 for adult uninjured zebrafish heart, N = 2 for 3 dpi and N = 2 for 7 dpi. Each sample for zebrafish hearts was a pool of n = 4–6 hearts. One-way ANOVA was performed to determine significantly changed metabolites.
To prepare the samples for GC/MS analysis, 30 uL of 20 mg/mL methoxyamine hydrochloride dissolved in pyridine was added to the dried aqueous metabolites, and samples were incubated at 37°C for 90 minutes. MTBSTFA (70 uL) was subsequently added and incubated at 37°C for 30 minutes. Samples were run on an Agilent 5977A Series GC/MSD system.
N = 2 for adult mouse heart, N = 3 for P7 mouse heart and N = 4 for P0.5 mouse heart.
N = 2 for adult uninjured zebrafish heart, N = 2 for 3 dpi and N = 2 for 7 dpi. Each sample for zebrafish hearts was a pool of n = 4–6 hearts. One-way ANOVA was performed to determine significantly changed metabolites.
5
GC/MS Analysis of Lemongrass Oil
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The components of lemongrass oil were analyzed by gas chromatography/mass spectrometry (GC/MS) using an Agilent 5977A Series GC/MSD System. Analytes were separated with a HP-5MS quartz capillary column (30 m x 0.25mm x 0.25μm). The analysis was performed using the following temperature program: initial temperature at 60°C, held for 2 min; and increased at the rate of 5°C /min to 90°C, held for 2 min; and increased at the rate of 5°C /min to 130°C; then increased at the rate of 10°C /min to 150°C, held for 1 min; and increased at the rate of 10°C /min to 200°C, held for 2 min; finally increased at the rate of 10°C /min to 240°C. Helium was used as the carrier gas (1 mL/min) at a split ratio of 200:1. The ion source temperature was 250°C, and mass spectra were obtained in EI-scan mode at 70 eV electron energy. The sector mass analyzer was set to scan from 35 to 500 amu. Identification of compounds was based on a comparison of mass spectra of each peak with those of authentic samples in a mass spectrum library. The percentages of compounds were calculated by the area normalization method.
6
Bioactive Compound Extraction and GC-MS Analysis
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ANU-301 was inoculation in 1 l of PDB medium for 5 days. After cultivation, broth medium was centrifuged at 10,000 ×g for 10 min and supernatant transferred to the conical flasks. In each conical flask, ethyl acetate were added to supernatant in a 1:1 ratio, shaken, and kept for overnight. Then, extraction solution were dried using a rotary evaporator. Extracts were dissolved in methanol for gas chromatography-tandem mass spectrometry (GC-MS/ MS) analysis and in DMSO for antifungal test. For GC-MS/MS analysis, 5977A Series GC/MSD System (Agilent Technologies, Santa Clara, CA, USA) was used. Temperature program of 40°C (1 min hold) to 300°C (10 min hold) at a rate of 10°C/min. The injector and mass interface temperature was 300°C, and the gas flow rate of the carrier gas, helium, was 1.0 ml/min. Injection mode was split and mass range was 50 to 400 (m/z). The result was analyzed using the National Institute of Standards and Technology spectral library version 11 (NIST 11 spectral library).
7
Characterization of Synthesized Nanoparticles
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The synthesized nanoparticles were analysed using Fourier transform infrared (FTIR) spectroscopy using a Bruker Vertex80 spectrophotometer in the wavenumber range 400 and 4000 cm−1. Samples were prepared in the form of pellets using KBr, maintaining the KBr : sample mass ratio at 1 : 10. Scanning electron microscopy (SEM) images and surface elemental analysis of AMNPs and AuPd@AMNPs was conducted using a Hitachi SU6600 FE-SEM. X-ray diffraction analysis of the synthesized AMNPs and AuPd@AMNPs was performed using a Bruker D8 Focus X-ray powder diffractometer using Cu Kα radiation (=0.154 nm) over the 2θ range of 5°–80°, with a step size of 0.02° and a step time of 1 s. Elemental analysis of AMNPs and AuPd@AMNPs was done using inductively coupled plasma mass spectrometry (ICPMS) using the Agilent 7000 ICPMS System. Samples: 25.0 mg were digested in H2O2 followed by dissolution in a mixture of HCl and HNO3, the final volume was made up to 10.0 ml. The products of catalytic reactions were characterized by GC–MS using Agilent 5977A Series GC/MSD System having a 30 m × 250 µm × 0.25 µm HP-5 ms column. The temperature programme was 40–250°C at 30°C min−1 with a final temperature isothermal hold for 12 min. The MS mass limit was set between 50 and 450 Da.
8
GC-MS Analysis of Fatty Acids
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The analyses were performed on the Agilent 5977A Series GC/MSD System (Stevens Creek Blvd.
Santa Clara, CA 95051, USA) as described by Durante et al. (2016) . Compounds were separated on DB-WAX column (60 m, 0.25 mm i.d., 0.25 mm film thickness, Agilent).The GC parameters were as follows: the temperature of the column was 50 °C after injection for 1 minute, then programmed at 25 °C/min to 200 °C, at 3 °C/min to 230 °C and maintained at constant temperature of 230 °C for 23 min. Split injection was conducted with a split ratio of 5:1, the flow-rate was 1.0 mL/min, carrier gas used was 99.999% pure helium, the injector temperature was 250 °C and the column head pressure was 40 psi for 0.4 min, constant pressure at 20 psi. The MS detection conditions were as follows: transfer line temperature 250°C, mode Scan, source and quadrupole temperature 230°C and 150°C respectively, scanning method of acquisition, ranging from 46 to 500, for mass/charge (m/z) was optimized. Spectrum data were collected at 0.5 s intervals. Solvent cut time was set at 2 min and 40 min retention time sufficient for separating all the fatty acids. Compounds were identified by using online NIST-library spectra and published MS data. Moreover, fatty acids standard were used to confirm MS data. A single technical replica was performed for each independent extract.
Santa Clara, CA 95051, USA) as described by Durante et al. (2016) . Compounds were separated on DB-WAX column (60 m, 0.25 mm i.d., 0.25 mm film thickness, Agilent).The GC parameters were as follows: the temperature of the column was 50 °C after injection for 1 minute, then programmed at 25 °C/min to 200 °C, at 3 °C/min to 230 °C and maintained at constant temperature of 230 °C for 23 min. Split injection was conducted with a split ratio of 5:1, the flow-rate was 1.0 mL/min, carrier gas used was 99.999% pure helium, the injector temperature was 250 °C and the column head pressure was 40 psi for 0.4 min, constant pressure at 20 psi. The MS detection conditions were as follows: transfer line temperature 250°C, mode Scan, source and quadrupole temperature 230°C and 150°C respectively, scanning method of acquisition, ranging from 46 to 500, for mass/charge (m/z) was optimized. Spectrum data were collected at 0.5 s intervals. Solvent cut time was set at 2 min and 40 min retention time sufficient for separating all the fatty acids. Compounds were identified by using online NIST-library spectra and published MS data. Moreover, fatty acids standard were used to confirm MS data. A single technical replica was performed for each independent extract.
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