The most active fungal extracts were selected to undergo secondary metabolites identification using gas-chromatography mass spectrometry GCMS, (GC-MS TQ8050; Shimadzu, Johannesburg, South Africa) equipped with a Multifunctional Autosampler (AOC-6000), a capillary column (RTX-5, 60 m × 0.25 mm × 0.25 µm, New Delhi, India) as described by Sharma et al. [7 ]. The identities of the compounds were determined by searching known molecules in databases of NIST05; WILEY 8, and FFNSC1.3 libraries.
Gcms tq8050
Manufactured by Shimadzu
Sourced in Japan
The GCMS-TQ8050 is a gas chromatograph-mass spectrometer (GC-MS) system manufactured by Shimadzu. It is designed for the analysis of a wide range of volatile and semi-volatile organic compounds. The GCMS-TQ8050 combines the separation capabilities of gas chromatography with the identification and quantification capabilities of triple quadrupole mass spectrometry.
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Lab products found in correlation
No 1 filter paper, by Cytiva (2 mentions)
Whatman, by Cytiva (2 mentions)
Aoc 6000, by Shimadzu (1 mentions)
Rtx 5, by Shimadzu (1 mentions)
Smart metabolites database, by Shimadzu (1 mentions)
Ms dial, by Shimadzu (1 mentions)
Zb 5ms plus, by Phenomenex (1 mentions)
Rxi 5ms capillary column, by Restek (1 mentions)
Hp 5ms, by Agilent Technologies (1 mentions)
Hp 5 capillary column, by Shimadzu (1 mentions)
Ftir 8400s spectrometer, by Shimadzu (1 mentions)
Agilent c18 column, by Agilent Technologies (1 mentions)
Spd 20a uv vis detector, by Shimadzu (1 mentions)
Q exactive focus orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Silica gel, by Qingdao Marine Chemical (1 mentions)
Sephadex lh 20, by GE Healthcare (1 mentions)
J 815 cd spectro polarimeters, by Jasco (1 mentions)
Gc 2010 plus, by Shimadzu (1 mentions)
12 protocols using gcms tq8050
1
Fungal Metabolite Identification by GC-MS
2
GC-MS/MS Metabolomic Profiling Protocol
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GC-MS/MS analysis was performed as previously described [49 (link)], using a GCMS-TQ8050 (Shimadzu Corporation, Kyoto, Japan). A 30 m × 0.25 mm (internal diameter) BPX-5 column (SGE, Melbourne, Australia) with a 0.25 µm film thickness was used, according to the method described in the Smart Metabolites Database (Shimadzu, Kyoto, Japan).
Data processing was performed using the Smart Metabolites Database (Shimadzu, Kyoto, Japan), MS-DIAL version 3.08 [50 (link)], and the MRMPROBS program version 2.42 [51 (link)]. Peaks were recorded for the 45−600 m/z mass range, and were automatically detected via MS-DIAL using the peak detection option of a minimum peak height of 2000. A data quality check was conducted using the thresholds of −10 < RI < 10, dot production > 0.8, and presence > 0.6, and the remaining data was then manually checked. Ultimately, 172 metabolites were identified in the plasma samples. The relative quantities of the metabolites were calculated using the peak areas of each metabolite relative to that of the internal standard (2-isopropylmalic acid), and expressed as a percentage of an arbitrary control set to 100%.
Data processing was performed using the Smart Metabolites Database (Shimadzu, Kyoto, Japan), MS-DIAL version 3.08 [50 (link)], and the MRMPROBS program version 2.42 [51 (link)]. Peaks were recorded for the 45−600 m/z mass range, and were automatically detected via MS-DIAL using the peak detection option of a minimum peak height of 2000. A data quality check was conducted using the thresholds of −10 < RI < 10, dot production > 0.8, and presence > 0.6, and the remaining data was then manually checked. Ultimately, 172 metabolites were identified in the plasma samples. The relative quantities of the metabolites were calculated using the peak areas of each metabolite relative to that of the internal standard (2-isopropylmalic acid), and expressed as a percentage of an arbitrary control set to 100%.
3
Pesticide Analysis in Livestock Products
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For the GC-MS/MS analysis of studied pesticides in livestock products, GCMS-TQ8050 (Shimadzu, Kyoto, Japan) was used. The columns used in the analysis were ZB-5MS Plus (30 m × 0.25 mm, 0.25 µm, Phenomenex Inc., Torrance, CA, USA). The carrier gas was helium at a flow rate of 1.5 mL/min, and the Q2 collision gas was argon. The interface temperature was set at 300 °C for the splitless injection of 2 µL. The oven temperature was initially set at 90 °C, which was maintained for 3 min; it was then increased to 120 °C at a rate of 20 °C/min; and then increased at a rate of 8 °C/min to 300 °C and maintained for 3 min. The GC-MS/MS analytical conditions are presented in detail in Table A2 .
4
GCMS-TQ8050 Gas Chromatography Protocol
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The GCMS-TQ8050 instrument from Shimadzu (Kyoto, Japan) was used with the Rxi-5MS capillary column (length, 20 mm; inner diameter, 0.18 mm; film thickness, 0.18 μm; Restek, Bellefonte, PA, USA) for gas chromatography mass spectrometry. The inlet temperature was 260°C, and the column flow rate was 1 mL per minute. The injection volume was 1 μL, and the splitless mode was used. During GCMS-TQ8050 analysis, the interface temperature was maintained at 250°C.
5
Glucose Quantification by GC-MS
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Twenty microliters per sample were added to glass vials with inserts to evaluate the glucose content via GC–MS (GC/MS-TQ8050, Shimadzu Inc.) using SIM mode and analytical conditions previously described44 . The glucose concentration in the samples was performed by comparing the obtained absorbance values in each sample with the glucose (Sigma) calibration curve.
6
GC-MS/MS Analysis of Pesticides in Greens
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For the analysis of 52 GC-amenable pesticides in green leafy vegetables, GC-MS/MS (GC-MS TQ8050, Shimadzu, Tokyo, Japan) with multiple reaction monitoring (MRM) acquisition mode was used. The column used for the chromatographic separation of analytes was an HP-5MS (30 m × 0.25 mm × 0.25 µm, Agilent Technologies, Palo Alto, CA, USA). High-purity helium at a constant flow rate of 2.4 mL min−1 was used as the carrier gas. Two microliters of the extract were injected in splitless mode. The column oven temperature was programmed at 50 °C, held for 0.0 min, ramped at 50 °C/min to 150°C, ramped at 20 °C/min to 230 °C and held for 1 min, and finally ramped at 8 °C/min to 290 °C, at which it was held for 18.5 min. The MS transfer line and ion source temperatures were sat at 280 and 200 °C, respectively. The MS operated in the electron impact ionization (EI) at 70 eV. Two ion transitions at the experimentally optimized collision energy were monitored for each target compound (Table S2 ). Both pairs of the MRM transitions were used for confirmation analysis, and the most sensitive transitions were selected for quantification analysis.
7
GC-MS Analysis of Volatile Compounds
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The samples (1.0 µL) were analyzed in random sequence, using Shimadzu GC–MS (TQ8050, Shimadzu Corporation, Tokyo, Japan). Shimadzu HP-5 capillary column (30 m × 250 µm × 0.25 µm) was used for the separation of GC compounds. High-purity helium was used as the carrier gas with a carrier gas flow rate of 1.0 mL min−1. The initial temperature of the column was set to 70 ℃, and maintained at this temperature for 4 min, and then increased to 300℃ at a rate of 8℃ min−1. The ionization mode was electron bombardment, the electron energy was 70 eV, and the temperature was 200 ℃. A mass range of 35 ~ 800 m z−1 was used for the mass spectra.
8
Spectroscopic Characterization of Compounds
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Optical rotations were recorded using a Rudolph Research Analytical automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). Circular dichroism (CD) was measured on a JASCO J-815 CD spectro polarimeters (JASCO Corporation, Tokyo, Japan). IR spectra were acquired by KBr disk method on a Shimadzu FTIR-8400S spectrometer (Shimadzu Co. Ltd., Tokyo, Japan). 1D and 2D nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV-500 spectrometer (Bruker Biospin Group, Karlsruhe, Germany) with TMS as an internal standard. Mass spectra were measured on Thermo Scientific Q Exactive Focus Orbitrap mass spectrometer (Waltham, MA, USA) for HRESIMS. GC-MS analysis was conducted by GCMS-TQ8050 (Shimadzu Co. Ltd., Tokyo, Japan) using the Rtx-5MS column (30 m × 0.25 mm × 0.25 μm).
Semi-preparative HPLC was conducted on an Agilent C18 column (250 × 10 mm I.D., 5 μm) equipped with a LC-6AD pump and a Shimadzu SPD-20A UV-Vis detector (Shimadzu Co. Ltd., Japan). Column chromatography (CC) was performed with silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical plant, Qing dao, China), ODS-C18 (50 μm, YMC Co. Ltd., Kyoto, Japan), and Sephadex LH-20 (18–110 μm, GE healthcare, Fairfield, CT, USA). Chromatographic or analytical grade solvents were used for isolation and separation procedures.
Semi-preparative HPLC was conducted on an Agilent C18 column (250 × 10 mm I.D., 5 μm) equipped with a LC-6AD pump and a Shimadzu SPD-20A UV-Vis detector (Shimadzu Co. Ltd., Japan). Column chromatography (CC) was performed with silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical plant, Qing dao, China), ODS-C18 (50 μm, YMC Co. Ltd., Kyoto, Japan), and Sephadex LH-20 (18–110 μm, GE healthcare, Fairfield, CT, USA). Chromatographic or analytical grade solvents were used for isolation and separation procedures.
9
Metabolomic Profiling of Murine CD4+ T Cells
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Isolated murine CD4+ T cells were subjected to gas chromatography–mass spectrometry (GC-MS) as described previously (77 (link)). Briefly, sonicated samples with 100% methanol were evaporated using a centrifugal evaporator (DNA120OP230, Thermo Fisher Scientific) and lyophilized (FD-1-84A, FTS Systems). Then, the samples were derivatized with methoxamine/pyridine and N-methyl-N-(trimethylsilyl)trifluoroacetamide and analyzed the GCMS-TQ8050 (Shimadzu). The chromatograms and mass spectra were evaluated with the GCMSsolution software 4.50 (Shimadzu). Compounds were determined with the Smart Metabolites Database Ver.2 (Shimadzu). The data obtained were then scaled (mean-centered and divided by the standard deviation of each variable) and analyzed using MetaboAnalyst (78 (link)).
10
Testicular Steroid Profiling in Mice
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Testes obtained from eight-week-old XY and XX/Sry mice were lyophilized using a Vacuum Centrifugal Evaporator (CVE-2000; EYELA, Tokyo, Japan) and stored at − 80 °C until later use. Gas chromatography–mass spectrometry steroid profiling was performed using a Shimadzu GC 2010 Plus gas chromatograph coupled to a triple-quadrupole GCMS-TQ8050 (Shimadzu Corporation, Kyoto, Japan) as previously described70 (link). Quantitative results were based on absolute quantities of steroid molecules per testis, and their metabolic ratios were also calculated to express their corresponding enzymatic activities.
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