The selected DPCMH24 fraction was analyzed by a Shimadzu GCMS-TQ8040 system (Japan). Capillary column; Rtx-5MS fused-silica, column length 30.0 m, internal diameter 0.25 μm. Sample injection; split mode (ratio 2) injector port temperature 280 °C. GC oven program; hold at 260.0 °C for 3 minutes, increase to 320.0 °C at 6.0 °C min−1, increasing to 330.0 at 5.0 °C min−1 and hold for 5 minutes. Carrier gas; helium at a constant flow rate (0.73 mL min−1).10 (link)
Gcms tq8040 system
Manufactured by Shimadzu
Sourced in Japan
The GCMS-TQ8040 system is a gas chromatograph-triple quadrupole mass spectrometer (GC-MS/MS) designed for high-sensitivity and high-selectivity analysis. The system combines a gas chromatograph with a triple quadrupole mass spectrometer, allowing for the precise identification and quantification of target compounds in complex matrices.
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7 protocols using gcms tq8040 system
1
GC-MS Analysis of DPCMH24 Fraction
2
GC-MS Analysis Parameters and Conditions
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The GC-MS parameters and conditions were similar to those used in the previous work [17 (link)]. A Shimadzu GCMS-TQ8040 system (Shimadzu Corporation, Kyoto, Japan) was used to perform GC-MS analysis. GC-MS parameters (polar stationary phase) were used as follows: an injection volume of 1 μL, a sample injection temperature of 250 °C, a split injection mode with a ratio of 1:5, and a carrier gas flow rate of 1.0 mL/min. Temperature program: 3 min at 50 °C, ramping the temperature to 250 °C at a rate of 8 °C/min, then 9 min at 250 °C; ion source temperature 200 °C and scanning range 40–500 m/z. The conditions used for a non-polar stationary phase were the same except for the carrier gas flow rate and temperature program—flow rate: 1.46 mL/min; program: 3 min at 50 °C, then ramping to 320 °C at a rate of 8 °C/min, then 5 min at 320 °C. The NIST 17 (NIST, Gaithersburg, MD, USA) database and the AMDIS software, version 2.3 (NIST, USA) were used for the library search and for deconvolution, respectively. Linear RIs based on n-alkanes were used. More details are provided in the previous work [17 (link)].
3
GC/MS Metabolite Extraction and Profiling
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After saline wash, cells were quenched by pouring liquid nitrogen into six-well plates and then harvested with ice-cold methanol:chloroform:scyllo-inositol (MeOH:CHCl3 9:1 v/v) containing 3 μM scyllo-inositol as internal standard. The extracts were vortexed for 10 s and incubated on ice for 15 min. By centrifugation at 4°C for 3 min at 16,100 g, the supernatant was collected and snap-frozen in liquid nitrogen and stored at –80°C.
The samples were evaporated to dryness by vacuum centrifugation. Prior to GC/MS analysis, samples were derivatized with 25 μl 3% (w/v) methoxyamine in pyridine (Sigma, #226904/270970) for 60 min at 37°C with mixing at 750 rpm, followed by trimethylsilylation with 25 μl BSTFA+1% TMCS (Thermo, #38831) for 60 min at 37°C with mixing at 750 rpm. The derivatized sample (1 μl) was analyzed using Shimadzu GC/MS-TQ8040 system, running the Shimadzu Smart Metabolites NRM database, comprising approximately 475 metabolite targets. Statistical analyses were performed using Student’s t-test following log transformation and median normalization. Metabolites were considered to be significant if their adjusted p-values after Benjamini-Hochberg correction were less than 0.05. Further data analysis and enrichment analysis were performed through MetaboAnalyst 4.0.
The samples were evaporated to dryness by vacuum centrifugation. Prior to GC/MS analysis, samples were derivatized with 25 μl 3% (w/v) methoxyamine in pyridine (Sigma, #226904/270970) for 60 min at 37°C with mixing at 750 rpm, followed by trimethylsilylation with 25 μl BSTFA+1% TMCS (Thermo, #38831) for 60 min at 37°C with mixing at 750 rpm. The derivatized sample (1 μl) was analyzed using Shimadzu GC/MS-TQ8040 system, running the Shimadzu Smart Metabolites NRM database, comprising approximately 475 metabolite targets. Statistical analyses were performed using Student’s t-test following log transformation and median normalization. Metabolites were considered to be significant if their adjusted p-values after Benjamini-Hochberg correction were less than 0.05. Further data analysis and enrichment analysis were performed through MetaboAnalyst 4.0.
4
Headspace-SPME Volatile Profiling of Flowers
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The volatile compounds were collected using a headspace-solid phase microextraction (HS-SPME)/GC-MS method. The whole flower was sealed in a 20-mL extraction bottle and equilibrated at 50°C for 10 min. The volatile compounds were extracted and adsorbed for 30 min using an SPME fiber [polydimethylsiloxane (PDMS), diameter 65 μm]. Then, the trapped floral scent compounds were analyzed using a Shimadzu GCMS-TQ8040 system. An Rxi-5Sil MS capillary column (30 m × 0.25 mm × 0.25 μm) was used for the separation of volatile compounds with helium as the carrier gas, at a flow rate of 1 mL/min. The column operating conditions were as follows: an initial 50°C hold step for 2 min, followed by an increase to 170°C at a rate of 4°C/min (held for 1 min), then to 270°C at a rate of 20°C/min, and a final hold step for 2 min. The volatiles were identified by comparing the mass spectra and retention times with authentic standards.
5
GC-MS Analysis of Chemical Compounds
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The GC-MS analysis was conducted using a Shimadzu GC-MS-TQ8040 system (Kyoto, Japan) coupled directly to a MS detector. The chromatographic separation was performed using the Rtx-5MS (5% phenyl-95% polydimethyl siloxane; 30 m × 0.25 mm ID, 0.25 μm) fused-silica column supplied by Restek (Bellefonte, United States). Helium was used as carrier gas at a constant flow rate of 1 L min–1. The ion source and interface temperatures were set at 230 and 290°C, respectively. Sample injection used a split mode (ratio 10:1) and the injector port temperature was set to 280°C whilst the GC oven temperature program was set as follows: 80°C (3 min hold), ramped to 110°C (5 min hold) at 10°C min–1, increased to 190°C (3 min hold), ramped to 220°C (4 min hold) at 10°C min–1, and then increased to 280°C (13 min hold) at 15°C min–1. The MS detector, operated at 70 eV, was set to scan from 60 to 665 atomic mass units. The compounds were identified by comparing spectra of the analyzed samples with the National Institute of Standards and Technology (NIST) library, 2008 and Wiley Registry of Mass Spectral Data, 8th Edition. High match scores (>95%) were considered for compound identification (Lee et al., 2016 (link)) and where possible this was checked with published literature.
6
Comprehensive Steroid Metabolome Analysis
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Steroid metabolome (95 steroids and their metabolites) was determined using a GCMS-TQ8040 system from Shimadzu (Kyoto, Japan) consisting of a gas chromatograph equipped with an automatic flow control, an AOC-20s autosampler and a triple quadrupole detector with an adjustable electron voltage of 10-195 V. The analysis was conducted in multiple reaction monitoring (MRM) mode. A capillary column with a medium polarity RESTEK Rtx-50 column (diameter 0.25 mm, length 15 m, film thickness 0.1 μm) was used for analyses. Electron-impact ionization with electron voltage fixed at 60 V and emission current set to 151 μA was used for the measurements (Table 1: GC) (Hill et al. 2018) . Selected steroids were detected by the method LC/MS/MS (Table 1: LC). Estrone (E1), estradiol (E2) and estriol (E3) were quantified using previously published LC-MS/MS method. Cortisol, cortisone, dehydroepiandrosterone (DHEA), 7α-hydroxydehydroepiandrosterone (7α-OH-DHEA), 7β-hydroxydehydroepiandrosterone (7β-OH-DHEA), 7-oxo-dehydroepiandrosterone (7-oxo-DHEA), pregnenolone, 17α-hydroxy-pregnenolone (17-OH-pregnenolone), testosterone, androstendione, progesterone, 17-OHprogesterone, corticosterone were determined using another LC-MS/MS validated method (Sosvorova et al. 2015b , Vitku et al. 2015) .
7
GCMS-TQ8040 Analysis of Organic Compounds
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The instrument used was a GCMS-TQ8040 system from Shimadzu (Kyoto, Japan) consisting of a gas chromatograph equipped with an automatic flow control, an AOC-20s autosampler and a triple quadrupole detector with an adjustable electron voltage of 10-195 V. The analysis was conducted in multiple reaction monitoring (MRM) mode. A capillary column with a medium polarity RESTEK Rtx-50 column (diameter 0.25 mm, length 15 m, film thickness 0.1 μm) was used for analyses. Electron-impact ionization with electron voltage fixed at 60 V and emission current set to 151 μA was used for the measurements. The temperatures of the injection port, ion source and interface were maintained at 220, 300, and 310 °C, respectively. Analyses were carried out in the splitless mode with a constant linear velocity of the carrier gas (He), which was maintained at 60 cm/s. The septum purge flow was set to 3 ml/min. The samples were injected using a high-pressure mode, which was applied at 200 kPa and maintained for 1 min. The detector voltage was set to 2.2 kV. The temperature program was as follows: 1 min delay at 80 °C, increase to 190 °C (40 °C/min), increase to 210 °C (6 °C/min), increase to 300 °C (20 °C/min), increase to 320 °C (40 °C/min), 4 min delay at 320 °C, initial pressure 34 kPa, injector temperature 220 °C, analysis duration 16.08 min.
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