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Bio bond c4 column

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The Bio-Bond C4 column is a chromatography column used for the purification and separation of biomolecules. It features a reversed-phase stationary phase with a C4 carbon chain ligand. The column is designed to provide high-resolution separation and efficient recovery of target analytes.

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Lab products found in correlation

Q exactive, by Thermo Fisher Scientific (3 mentions) Ultimate 3000lc, by Thermo Fisher Scientific (1 mentions) Exactive plus orbitrap, by Thermo Fisher Scientific (1 mentions)

5 protocols using bio bond c4 column

1

Quantification of Serum Choline and TMAO

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Serum choline and TMAO levels were measured according published methods [5 (link)]. In brief, serum samples were prepared for analysis by precipitating proteins with 4 volumes of ice-cold methanol spiked with 2.5 μM deuterium-labeled choline and deuterium-labeled TMAO internal standards. Samples were centrifuged at 18,213×g at 4 °C for 3 min. The recovered supernatants were diluted 1:1 in uHPLC-grade water prior to screening. After sample preparation, identification and quantitation of TMAO and choline were performed using a uHPLC (Thermo Scientific/Dionex 3000) coupled to a high-resolution mass spectrometer (Thermo Scientific Q Exactive). Liquid chromatography separation was achieved on a Dikma Bio-Bond C4 column (150 mm × 2.1 mm; 3-μm particle size) using a 7 min isocratic gradient (50:50 methanol [MeOH] − water, 5 mM ammonium formate, and 0.1% formic acid). Quantitation of TMAO (76.0762) and d9-TMAO (85.1318) was performed via targeted MS/MS in positive mode using the following fragments masses: TMAO (58.0659) and d9-TMAO (68.1301). Quantitation of choline (104.1075) and d9-choline (113.1631) was performed in positive mode with full-MS scan by monitoring their exact masses.
Romano K.A., Dill-McFarland K.A., Kasahara K., Kerby R.L., Vivas E.I., Amador-Noguez D., Herd P, & Rey F.E. (2018). Fecal Aliquot Straw Technique (FAST) allows for easy and reproducible subsampling: assessing interpersonal variation in trimethylamine-N-oxide (TMAO) accumulation. Microbiome, 6, 91.
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2

Quantitative Analysis of Hemoglobin Globin Chains

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Samples were prepared from hemolysate with an Hb concentration of 40 g/L and were diluted further with water (75 μL hemolysate plus 925 μL water). Total 20 μL of diluted samples were used for each assay. Bio-Bond C4 column (5 μm, 250 x 4.6 mm, DIKMA) was used for RP-HPLC analysis. We eluted globin chains with a two-solvent system [solvent A, 200 mL/L acetonitrile and 3 mL/L trifluoroacetic acid (TFA) in water; solvent B, 600 mL/L acetonitrile and 3 mL/L TFA in water] and a 3-step RPLC elution program consisting of a linear gradient of 60%–100% solvent B in 80 min, a linear gradient of 100%–60% solvent B in 10 min, and reequilibration with 60% solvent B for 10 min. The flow rate was 1 mL/min, eluate was detected at 220 nm [38 (link)] and abundance were quantified using Image J [39 ]. Molecular weight of globins were detected using a MaXis 4G ultra-high resolution time of flight mass spectrometer (Bruker-Daltonics).
Lu S., Xin Y., Tang X., Yue F., Wang H., Bai Y., Niu Y, & Chen Q. (2015). Differences in Hematological Traits between High- and Low-Altitude Lizards (Genus Phrynocephalus). PLoS ONE, 10(5), e0125751.
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3

Quantification of Choline and TMAO in Plasma

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Plasma samples were prepared for analysis by precipitating proteins with 4 volumes of ice-cold methanol spiked with 2.5 μM deuterium-labeled choline and TMAO internal standards. Samples were centrifuged at 18,213 × g at 4°C for 3 min. The recovered supernatants were diluted 1:1 in uHPLC-grade water prior to screening. Identification and quantification of choline and TMAO was performed using a uHPLC (Dionex 3000) coupled to a high-resolution mass spectrometer (Thermo Scientific Q Exactive). Liquid chromatography separation was achieved on a Dikma Bio-Bond C4 column (150 mm by 2.1 mm; 3-μm particle size) using a 7-min isocratic gradient (50:50 methanol-water, 5 mM ammonium formate, and 0.1% formic acid). A heated electrospray ionization interface, working in positive mode, was used to direct column eluent to the mass spectrometer. Quantitation of TMAO and D9-TMAO was performed via targeted MS/MS using the following paired masses of parent ions and fragments: TMAO (76.0762 and 58.0659) and D9-TMAO (85.1318 and 68.1301). Quantitation of choline and d9-choline was performed in full-MS scan mode by monitoring their exact masses: 104.1075 and 113.1631, respectively.
Kasahara K., Kerby R.L., Zhang Q., Pradhan M., Mehrabian M., Lusis A.J., Bergström G., Bäckhed F, & Rey F.E. (2023). Gut bacterial metabolism contributes to host global purine homeostasis. Cell host & microbe, 31(6), 1038-1053.e10.
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4

Quantitative Analysis of Choline Metabolites

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After sample preparation, identification and quantitation of TMA, TMAO, and choline was performed using a uHPLC (Dionex 3000) coupled to a high-resolution mass spectrometer (Thermo Scientific Q Exactive). Liquid chromatography separation was achieved on a Dikma Bio-Bond C4 column (150 mm by 2.1 mm; 3-µm particle size) using a 7-min isocratic gradient (50:50 methanol [MeOH]−water, 5 mM ammonium formate, and 0.1% formic acid). A heated electrospray ionization interface, working in positive mode, was used to direct column eluent to the mass spectrometer. Quantitation of TMA, D9-TMA, TMAO, and D9-TMAO was performed via targeted MS/MS using the following paired masses of parent ions and fragments: TMA (146.118 and 118.0865), D9-TMA (155.1740 and 127.1434), TMAO (76.0762 and 58.0659), and D9-TMAO (85.1318 and 68.1301). Quantitation of choline and d9-choline was performed in full-MS scan mode by monitoring their exact masses: 104.1075 and 113.1631, respectively.
Romano K.A., Vivas E.I., Amador-Noguez D, & Rey F.E. (2015). Intestinal Microbiota Composition Modulates Choline Bioavailability from Diet and Accumulation of the Proatherogenic Metabolite Trimethylamine-N-Oxide. mBio, 6(2), e02481-14.
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5

Lipid Profiling by High-Resolution LC-MS

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Lipid samples were analyzed at the Harvard Center for Mass Spectrometry. The LC–MS analyses were modified from Miraldi81 (link) and were performed on an Orbitrap Exactive plus (Thermo Scientific) in line with an Ultimate 3000 LC (Thermo Scientific). Each sample was analyzed in positive and negative modes, in top 5 automatic data-dependent MSMS mode. Column hardware consisted of a Biobond C4 column (4.6 × 50 mm, 5 μm, Dikma Technologies). Flow rate was set 100 μl min−1 for 5 min with 0% mobile phase B (MB), then switch to 400 μl min−1 for 50 min, with a linear gradient of MB from 20 to 100%. The column was then washed at 500 μl min−1 for 8 min at 100% MB before being reequilibrated for 7 min at 0% MB and 500 μl min−1. For positive mode runs, buffers consisted for mobile phase A (MA) of 5 mM ammonium formate, 0.1% formic acid and 5% methanol in water, and for mobile phase B (MB) of 5 mM ammonium formate, 0.1% formic acid, 5% water, 35% methanol in isopropanol. For negative runs, buffers consisted for MA of 0.03% ammonium hydroxide, 5% methanol in water, and for MB of 0.03% ammonium hydroxide, 5% water, 35% methanol in isopropanol. Lipids were identified and quantified using the Lipidsearch© software (version 4.2.27, Mitsui Knowledge Industry, University of Tokyo). Integrations and peak quality were curated manually before exporting and analyzing the data in Microsoft Excel.
Bopp S., Pasaje C.F., Summers R.L., Magistrado-Coxen P., Schindler K.A., Corpas-Lopez V., Yeo T., Mok S., Dey S., Smick S., Nasamu A.S., Demas A.R., Milne R., Wiedemar N., Corey V., Gomez-Lorenzo M.D., Franco V., Early A.M., Lukens A.K., Milner D., Furtado J., Gamo F.J., Winzeler E.A., Volkman S.K., Duffey M., Laleu B., Fidock D.A., Wyllie S., Niles J.C, & Wirth D.F. (2023). Potent acyl-CoA synthetase 10 inhibitors kill Plasmodium falciparum by disrupting triglyceride formation. Nature Communications, 14, 1455.
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