Agilent 5975 mass selective detector

Manufactured by Agilent Technologies

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The Agilent 5975 mass selective detector is a standalone gas chromatography-mass spectrometry (GC-MS) system designed for analyte identification and quantification. It features an electron ionization (EI) source and a quadrupole mass analyzer to detect and analyze compounds eluted from a gas chromatograph.

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9 protocols using agilent 5975 mass selective detector

Carbon Fixation and Fatty Acid Analysis in Microalgae

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C-169 cells from 0.04 % CO₂ and 2 % CO₂ were collected on the 4th, 8th and 12th day and lyophilized into cell pellets. Total carbon content (C_C, % dry cell weight) was analyzed by an element analyzer (EuroEA3000, EuroVector S.p.A., Italy). CO₂ fixation rate (

R_{{CO}_{2}}

, g L⁻¹ day⁻¹) was determined as previously described [49 (link)]. It was calculated using the following equation:

R_{{CO}_{2}}

= C_CP(

M_{{CO}_{2}}

/Mc), where P is the biomass productivity (g L⁻¹ day⁻¹), M_C is the molecular weight of carbon, and

M_{{CO}_{2}}

is the molecular weight of CO₂.
Fatty acid profiling was performed on the lyophilized cell pellets (Modul YOD-230, Thermo-Fisher, USA) via gas chromatography mass spectrometry (Agilent 6890 gas chromatography coupled with Agilent 5975 mass selective detector, Agilent Technologies, Santa Clara, CA, USA). Nonadecanoic acid (C19:0, Sigma-Aldrich, St. Louis, MO, USA) was added as internal standard to quantify FA content. Fatty acid methyl esters (FAMEs) were prepared and analyzed according to the protocol as previously described [50 (link)]. The degree of lipid unsaturation (DLU) was calculated according to previously described [51 (link)]:

DLU (▿ / mole) = [1.0 \times (% monoene) + 2.0 \times (% diene) + 3.0 \times (% triene)] / 100 .

Peng H., Wei D., Chen G, & Chen F. (2016). Transcriptome analysis reveals global regulation in response to CO2 supplementation in oleaginous microalga Coccomyxa subellipsoidea C-169. Biotechnology for Biofuels, 9, 151.

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Quantifying α-Ketoglutarate Enrichment

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Cells were cultured in DMEM containing [U-¹³C]glutamine and extracts were collected in ice cold 1:1 methanol:water, subjected to three freeze-thaw cycles, and cell debris removed by high-speed centrifugation with cold 80% aqueous methanol^{45 (link)}. Subsequently, the supernatant was split into two glass tubes and evaporated with blown air at 42°C. To one of the dried samples, 25 nmoles of unlabelled α-KG was added and the sample was evaporated to dryness again. To all samples, 50 µL of methoxyamine-hydrochloride in pyridine (2%) was added and the samples were left at room temperature overnight (~16h). The samples were then evaporated again and derivatized in 100 µL Tri-Sil reagent (Thermo) at 42°C for 1.5h. All samples were then injected onto an Agilent 6890N gas chromatograph networked to an Agilent 5975 Mass Selective Detector. Mass isotopomer distribution was determined for α-KG using methods analogous to those used for other TCA cycle intermediates^{45 (link)}. The abundance of α-KG was calculated based on the fold dilution of enrichment caused by addition of 25 nmoles unlabeled α-KG.

Lin A.P., Abbas S., Kim S.W., Ortega M., Bouamar H., Escobedo Y., Varadarajan P., Qin Y., Sudderth J., Schulz E., Deutsch A., Mohan S., Ulz P., Neumeister P., Rakheja D., Gao X., Hinck A., Weintraub S.T., DeBerardinis R.J., Sill H., Dahia P.L, & Aguiar R.C. (2015). D2HGDH regulates alpha-ketoglutarate levels and dioxygenase function by modulating IDH2. Nature Communications, 6, 7768.

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GC/MS Analysis of PBMC Metabolites

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GC/MS analysis was carried out according to this group’s previously published work [7 (link), 30 (link)]. Briefly, each 1 μL of the derived sample was injected into an Agilent 7890A GC system (Agilent Technologies Inc., USA). An HP-5 MS fused silica capillary column (30 m × 0.25 mm × 0.25 μm, Agilent, USA) was used for metabolite separation with helium carrier gas at a flow rate of 1 mL/min. The injector temperature was set at 280°C. The column temperature was initially kept at 80°C for 2 min and then increased to 320°C at 10°C/min, where it was held for 6 min. The column effluent was introduced into the ion source of an Agilent 5975 mass selective detector (Agilent Technologies). The MS quadrupole temperature was set at 150°C, and the ion source temperature was set at 230°C. Data acquisition was performed first in the full-scan mode (scanning range from 50 to 550 m/z) and then in selected ion monitoring (SIM) mode for quantification. The characteristic fragment ions and retention times of metabolites were shown in Additional file 1: Table S1. All samples were analyzed consecutively at random. A quality control (QC) sample, pooled from a representative PBMCs sample of each group, was added in each batch of analyses in order to adjust the variations between batch variability.

Liu M.L., Zhang X.T., Du X.Y., Fang Z., Liu Z., Xu Y., Zheng P., Xu X.J., Cheng P.F., Huang T., Bai S.J., Zhao L.B., Qi Z.G., Shao W.H, & Xie P. (2015). Severe disturbance of glucose metabolism in peripheral blood mononuclear cells of schizophrenia patients: a targeted metabolomic study. Journal of Translational Medicine, 13, 226.

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Metabolite Analysis by GC-MS

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Five-gram subsamples were added to 10 ml ddH₂O, placed in an ultrasonic cleaner (AS30600B; Autoscience, Tianjin, China) for 30 min, and centrifuged at 8,000 × g for 10 min. After filtering using an 0.2-μm-pore-size filter, the filtrate was used to analyze metabolite concentrations (27 (link)). Metabolites were detected using gas chromatography-mass spectrometry (Agilent 6890N GC system and Agilent 5975 mass selective detector; Agilent, Santa Clara, CA) with condition details based on a previous study (65 (link)).

Wang S., Xiong W., Wang Y., Nie Y., Wu Q., Xu Y, & Geisen S. (2020). Temperature-Induced Annual Variation in Microbial Community Changes and Resulting Metabolome Shifts in a Controlled Fermentation System. mSystems, 5(4), e00555-20.

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GC-MS Analysis of Volatile Compounds in Lily Rice Wine

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Agilent 6890 GC equipped with an Agilent 5975 mass selective detector (Agilent, USA) was used. Helium was used as the carrier gas at a constant flow rate of 1 mL/min. The temperature of the injector and detector were both set at 250 °C. The analytes were separated through a DB-Wax column (60 m length, 0.25 mm i.d., 0.25 mm film thickness, Agilent) with an oven temperature program of 40 °C (1 min), 5 °C/min to 180 °C (1 min), and 8 °C/min to 230 °C (7 min). The Agilent 5975 MSD was used for identifying the unknown compounds. The electron impact energy was 70 eV, and the ion source and quadruple temperatures were both set at 230 °C. Electron impact (EI) mass spectra was recorded in the 20–550 amu range. All samples were analyzed in triplicates. The mass spectrum data were compared against NIST mass spectral database (Agilent Technologies Inc.) to identify the volatile flavor compounds in lily rice wine samples. The volatile compounds were quantified by comparing their peak areas to that of the 2-octanol internal standard. The amounts of individual constituents present in the sample were calculated and expressed as milligram per liter of wine.

Yan S., Xiangsong C, & Xiang X. (2019). Improvement of the aroma of lily rice wine by using aroma-producing yeast strain Wickerhamomyces anomalus HN006. AMB Express, 9, 89.

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Isotopic Tracing of Glucose and Lactate Metabolism

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HCT116 and LS174T cells were seeded into six-well plates at a density of 6 × 10⁵ cells per well and incubated for 24 h. For glucose-labelling experiments media were supplemented with glutamine (2 mM), lactic acid (10 mM) and ¹³C₆-glucose (5.6 mM, Sigma-Aldrich, Missouri, US). For lactic acid-labelling experiments media were supplemented with glutamine (2 mM), glucose (5.6 mM) and ¹³C₃-lactic acid (10 mM, Sigma-Aldrich, Missouri, US). Media were also supplemented with uprosertib (10 μM) or vehicle (DMSO, 0.1%). Cells were treated for 4 h before intracellular metabolites were extracted and aqueous fractions were analysed using the Agilent 7890 GC system linked to an Agilent 5975 Mass Selective Detector using methods published previously.^{26 (link)} AMDIS software was used with reference to the NIST mass spectral library²⁷ to identify metabolites. Peak integration was done using in-house developed GAVIN^{28 (link)} scripts for MATLAB® (MathWorks).

Barnes E.M., Xu Y., Benito A., Herendi L., Siskos A.P., Aboagye E.O., Nijhuis A, & Keun H.C. (2020). Lactic acidosis induces resistance to the pan-Akt inhibitor uprosertib in colon cancer cells. British Journal of Cancer, 122(9), 1298-1308.

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Analyzing Ant Food Body Chemistry

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To compare acceptance of FB by ants in relation to differences in FB chemical profiles, external food body chemicals were extracted and chemical profiles were compared across host plant species. Chemical cues from ants were also extracted to describe any potential contamination of the chemicals present on the FB. We extracted a minimum of 10 FB and five ants per sample from 10 trees for each plant species. Samples for chemical cues were placed in 1.5‐ml glass vials with 1.5 ml of hexane for 10 min. The hexane was then transferred to another 1.5‐ml glass vial. Extracts were shipped to the University of Würzburg and were analyzed with an Agilent 6890 gas chromatograph coupled with an Agilent 5975 Mass Selective Detector (Agilent). For further details, see Appendix S2.

Houadria M.Y., Barone G., Fayle T.M., Schmitt T., Konik P, & Feldhaar H. (2023). An experimental, behavioral, and chemical analysis of food limitations in mutualistic Crematogaster ant symbionts inhabiting Macaranga host plants. Ecology and Evolution, 13(2), e9760.

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Characterization of PCB Sulfate Intermediates

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Melting points were measured on a Mel-Temp melting point apparatus and are uncorrected. If not stated otherwise, all PCB sulfates and the corresponding intermediates, were characterized by ¹H and ¹³C NMR spectroscopy. The NMR spectra were recorded on a Bruker Avance DRX-400 spectrometer in the University of Iowa Central NMR Research Facility (Iowa City, IA, USA). The NMR samples were prepared in CDCl₃ or CD₃OD (Cambridge Isotope Laboratories, Andover, MA), and tetramethylsilane (TMS) was used as internal standard. GC-MS analysis of TCE-PCB sulfates diester intermediates was performed in the electron impact (EI) mode on an Agilent 6890N gas chromatograph coupled with an Agilent 5975 Mass Selective Detector (Agilent Technologies, CA, USA) as reported previously (Telu et al. 2010 (link)). Only the isotopic ion with the lowest mass is reported for major fragments observed in the MS spectra. Accurate mass determinations of the PCB sulfates were performed by the High Resolution Mass Spectrometry Facility of the University of California Riverside (Riverside, CA, USA). The purity of all PCB sulfates was verified by thin layer chromatography as described previously (Li et al. 2010 (link)) prior to preparing DMSO stock solutions for cell culture experiments to ensure that no hydrolysis to the corresponding OH-PCB had occurred.

Flor S., He X., Lehmler H.J, & Ludewig G. (2015). Estrogenicity and androgenicity screening of PCB sulfate monoesters in human breast cancer MCF-7 cells. Environmental science and pollution research international, 23(3), 2186-2200.

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Spectrophotometric Analysis of Chlorophyll a and Lignin Phenols

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Chlorophyll a was measured spectrophotometrically (Hewlett-Packard photo-diode array, model 8453) using EPA method 446 and Lorenzen’s spectrophotometric equations^{28 ,29 (link)}. Copepod and particulate lignin analyses were performed using CuO oxidation as in Hedges & Ertel^{30 (link)} with modifications outlined in Spencer et al.³¹ and Hernes et al.^{32 (link)}. Briefly, samples were oxidized in 8% NaOH in the presence of excess CuO at 155 °C followed by acidification and ethyl acetate extraction. The extracted fraction was dried under a gentle stream of ultrapure nitrogen and trimethylsilyl derivitized with bis(trimethylsilyl)trifluoroacetamide (BSTFA) after redissolution in pyridine. Lignin phenols were separated using an Agilent 6890 gas chromatograph fitted with a DB5-MS capillary column (30 m, 0.25 mm inner diameter, J&W Scientific) and attached to an Agilent 5975 mass selective detector. Quantification was achieved using selective ion monitoring, standardization to an internal standard (cinnamic acid), and a five point calibration scheme of Hernes et al.^{33 (link)}. Samples were blank-corrected due to trace lignin in reagents.

Harfmann J., Kurobe T., Bergamaschi B., Teh S, & Hernes P. (2019). Plant detritus is selectively consumed by estuarine copepods and can augment their survival. Scientific Reports, 9, 9076.

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