Cholesterol, noncholesterol sterol, and oxysterol content was analyzed in macrophages and Jurkat cells by gas chromatography (GC). Cell pellets were spun in a speedvac concentrator (12 mbar; Savant AES 1000) and weighed. Cholesterol, noncholesterol sterols, and oxysterols were extracted using chloroform. After alkaline hydrolysis, the concentrations of cholesterol precursors were measured with GC-mass spectrometry-selected ion monitoring [19 (link)]. The trimethylsilyethers of the sterols were separated on a DB-XLB (30 m length × 0.25 mm internal diameter, 0.25 μm film) column (Agilent Technologies, Waldbronn, Germany) using the 6890N Network GC system (Agilent Technologies). Epicoprostanol (Steraloids, Newport, RI, USA) was used as an internal standard, to quantify the noncholesterol sterols (Medical Isotopes, Pelham, NH, USA) on a 5973 Network MSD (Agilent Technologies). Total cholesterol was measured by GC-flame ionization detection on an HP 6890 GC system (Hewlett Packard, Waldbronn, Germany), equipped with a DB-XLB (30 m length × 0.25 mm internal diameter, 0.25 μm film) column (Agilent Technologies) using 5a-cholestane (Steraloids) as internal standard [20 ].
Db xlb
Manufactured by Agilent Technologies
Sourced in Germany, United States
The DB-XLB is a capillary column designed for gas chromatography (GC) analysis. It is a highly inert, low-bleed column that provides excellent separation and peak shape for a wide range of analytes. The column is made of fused silica and coated with a proprietary stationary phase, making it suitable for a variety of applications.
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Lab products found in correlation
Hp6890 gc system, by Hewlett-Packard (2 mentions)
6890n network gc system, by Agilent Technologies (2 mentions)
5973 network msd, by Agilent Technologies (2 mentions)
Epicoprostanol, by Steraloids (2 mentions)
Db 17ms column, by Agilent Technologies (2 mentions)
5975b mass selective detector, by Agilent Technologies (1 mentions)
Db 5ms column, by Agilent Technologies (1 mentions)
Single quadrupole gcms qp2020 nx gas chromatograph mass spectrometer, by Shimadzu (1 mentions)
Combipal autosampler, by CTC Analytics (1 mentions)
6890 gas chromatograph, by Agilent Technologies (1 mentions)
8 protocols using db xlb
1
Quantifying Sterols in Macrophages and Jurkat Cells
2
Quantification of Cellular Sterols and Oxysterols
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The cellular content of cholesterol was measured by gas chromatography-flame ionization detection (GC-FID) and of non-cholesterol sterols and oxysterols by gas chromatography-mass spectrometry in selected ion monitoring mode (GC-MS-SIM). Cell pellets were spun in a speedvac concentrator (12 mbar; Savant AES 1000) and weighed. Cholesterol, non-cholesterol sterols, and oxysterols were extracted using chloroform. After alkaline hydrolysis, the concentrations of cholesterol precursors were measured with GC-MS-SIM as previously described [5 (link),87 (link)]. The trimethylsilyethers of the (oxy)sterols were separated on a DB-XLB (30 m length × 0.25 mm internal diameter, 0.25 μm film) column (Agilent Technologies) using the 6890 N Network GC system (Agilent Technologies). Epicoprostanol (Steraloids, Newport, RI, USA) was used as an internal standard, to quantify the non-cholesterol sterols (Medical Isotopes, Pelham, NH, USA) on a 5973 Network MSD (Agilent Technologies). Total cholesterol was measured by GC-FID on an HP 6890 GC system (Hewlett Packard, Waldbronn, Germany), equipped with a DB-XLB (30 m length × 0.25 mm internal diameter, 0.25 μm film) column (Agilent Technologies) using 5α-cholestane (Steraloids) as internal standard [32 (link),88 (link)].
3
GC-ECD and GC-MS Analysis of PSD and OSU Extracts
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PSD extracts were analysed for 44 compounds including PRCs, internal standards and internal surrogates using an Agilent 6890N gas chromatograph (GC) with dual 7683 injectors, dual columns and dual electron capture detectors (ECD). The GC was configured with Agilent DB-XLB and Agilent DB-17MS columns. In addition to quantitative evaluation, OSU extracts were also screened for the presence/absence of over 1100 chemicals of concern (see electronic supplementary material, table S2) on an Agilent 6890N GC with 5975B Mass Selective Detector with a DB-5MS column. Limits of quantitation (LOQ) are detailed in the electronic supplementary material.
4
Multi-Pesticide Analysis in Environmental Samples
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A total of 47 pesticides or pesticide degradation products were measured: 27 are classified as insecticides, 7 herbicides, 7 fungicides, and 6 pesticide degradation products. The method reflects our interest in insecticides for their potential human health impacts and includes 21 organochlorine, 3 organophosphate, and 3 pyrethroid insecticides. Pesticides were analyzed on an Agilent 6890N GC with dual 7683 injectors, dual columns, DB-XLB and DB-17MS columns (Agilent), and dual microelectron capture detectors (μ-ECD).47 (link)
5
GC-MS Analysis of Volatile Compounds
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GC-MS measurements were performed on a Shimadzu single quadrupole GCMS-QP2020 NX gas chromatograph–mass spectrometer (Shimadzu, Kyoto, Japan). Static headspace injection was carried out with a CombiPAL autosampler (CTC Analytics AG, Zwingen, Switzerland). After sample incubation at 75 °C for 20 min, 1 mL headspace volume of sample was injected with a gastight 2.5 mL heatable syringe (Hamilton, Reno, NV, USA) into the GC injector operated at 200 °C in split mode with a split ratio of 1:10. The syringe was heated constantly at 80 °C and flushed for 3 min before each injection cycle to avoid condensation effects and carry-over. The GC was equipped with a low-polarity capillary column (DB-XLB, 30 m × 0.25 mm × 0.25 μm, Agilent Technologies, Santa Clara, CA, USA) and operated with a column gas flow of 0.95 mL min−1 of helium. At 40 °C initial temperature, the oven program started, followed by a temperature ramp of 10 °C min−1 to 140 °C, at which point it was increased by 20 °C min−1 to 200 °C, resulting in a total run time of 13 min. The analytical column’s output was fed into an MS, equipped with an electron impact (EI) ion source operated at 230 °C.
6
GC-μECD Analysis of Environmental Samples
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An Agilent 6890 Gas Chromatograph equipped with dual micro electron capture detectors with dual columns Agilent DB-XLB (60 m, ID 0.25 mm, Film thickness 0.25 μm), SGE BPX-35 (60 m, ID 0.25 mm, Film thickness 0.25 μm) was used to perform the analysis. Hydrogen carrier gas was used at a flowrate of 1 ml/min with nitrogen makeup gas at a flowrate of 60 ml/min. Temperature program used was started at an initial temperature of 90°C then increased at 25°C/min to 280°C and held for 8.4 mins. The total run time was16 mins. The injection volume used was 1 μl splitless at 250°C.
7
Quantification of Residual PCBs in Sediments
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The analysis was done in an Agilent Technologies 6890N gas chromatograph (Agilent, Palo Alto, CA, USA) coupled to a Micromass AutoSpec—Ultima NT (Waters, Manchester, UK) high-resolution mass spectrometer (EBE geometry) controlled by a Masslynx data system.
The chromatographic system was equipped with a DB-XLB (Agilent, Folsom, CA, USA) fused-capillary column (60 m × 0.25 mm I.D. × 0.25 μm film thickness). Following the temperature program that can be seen elsewhere [14 (link)]. The injection of 1 µL of extract was carried out in splitless mode (60 s) with the temperature of the injector at 280 °C.
The chromatograph was coupled to a magnetic sector spectrometer equipped with an electron impact ionisation (EI) source working at 32 eV, trap current at 500 µA, and acceleration voltage at 8000 V. The acquisition was performed in selected ion monitoring (SIM) mode at a resolving power of 10,000 (5% valley). The ion source and transfer line were set at 250 and 280 °C, respectively.
The quantification of residual PCBs in sediments was done by isotopic dilution methodology described elsewhere [14 (link)].
The chromatographic system was equipped with a DB-XLB (Agilent, Folsom, CA, USA) fused-capillary column (60 m × 0.25 mm I.D. × 0.25 μm film thickness). Following the temperature program that can be seen elsewhere [14 (link)]. The injection of 1 µL of extract was carried out in splitless mode (60 s) with the temperature of the injector at 280 °C.
The chromatograph was coupled to a magnetic sector spectrometer equipped with an electron impact ionisation (EI) source working at 32 eV, trap current at 500 µA, and acceleration voltage at 8000 V. The acquisition was performed in selected ion monitoring (SIM) mode at a resolving power of 10,000 (5% valley). The ion source and transfer line were set at 250 and 280 °C, respectively.
The quantification of residual PCBs in sediments was done by isotopic dilution methodology described elsewhere [14 (link)].
8
Quantitative GC-MS/MS Analysis of Compounds
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Gas chromatography–tandem mass spectrometry (Agilent Technologies 7890GC/7000B, CA, USA) was employed. A DB-XLB (length 30 m, thickness 0.25 µm, and inner diameter 0.25 mm) capillary column (Agilent Technologies, CA, USA) was employed for the separation. The injection mode was pulsed splitless, its volume was 2.0 µL, and its temperature was 280 °C [12 (link)]. The temperature program of the oven was set at 80 °C for 2 min, ramped 20 °/min to 200 °C, ramped 10 °C /min to 300 °C, hold at 300 °C for 5 min, ramped 25 °C/min to 325 °C, and held at 325 °C for 11 min. Further, electron ionization (EI) and multiple reaction monitoring (MRM) modes were employed for quantitation. The EI voltage was 70 eV, and the MRM conditions are given in Table 1 . MRM is a combination of the precursor and productions. The compound was quantified by MRM1, employing the peak area value. The ratio of the peak area value of MRM2 to that of MRM1 was qualitatively compared to the standard solution.
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