Fecal metabolites were extracted from fresh thawed fecal samples (about 10 mg) suspended in 400 μL of 50% methanol in Millli-Q water supplemented with the internal standards (20 μM each of methionine sulfone and D-camphor-10-sulfonic acid (CSA)). The mixture was combined with two 3-mm zirconia beads (BioSpec Products, Bartlesville, OK, USA) and about 100 mg of 0.1-mm zirconia/silica beads (BioSpec Products, Bartlesville, OK, USA) and subjected to 3 min of vigorous shaking using a Micro Smash (TOMY, Nerima, Tokyo, Japan). The suspension was centrifuged at 4600× g for 15 min at room temperature, and the resulting supernatant was transferred to a 5-kDa-cutoff filter column (Ultrafree MC-PLHCC 250/pk for Metabolome Analysis, Human Metabolome Technologies, Tsuruoka, Yamagata, Japan). The flow-through was dried under vacuum and the residue then was dissolved in 40 μL of Milli-Q water containing reference compounds (200 μM each of 3-aminopyrrolidine and trimesate). The levels of extracted metabolites were measured in both positive and negative modes by CE-TOFMS as previously described [74 (link)]. All CE-TOFMS experiments were performed using an Agilent capillary electrophoresis system (Agilent Technologies, Santa Clara, CA, USA).
3 mm zirconia beads
Manufactured by Biospec
Sourced in United States
The 3-mm zirconia beads are hard, chemically inert, and durable spherical particles made from zirconium oxide. They are designed for use in a variety of laboratory applications, such as sample preparation, homogenization, and dispersal.
Automatically generated - may contain errors
Lab products found in correlation
Ultrafree mc plhcc 250 pk for metabolome analysis, by Human Metabolome Technologies (1 mentions)
Agilent capillary electrophoresis system, by Agilent Technologies (1 mentions)
0.1 mm zirconia silica beads, by Biospec (1 mentions)
Micro smash, by TOMY (1 mentions)
Agilent ce tofms system, by Agilent Technologies (1 mentions)
Methionine sulfone, by Fujifilm (1 mentions)
D camphor 10 sulfonic acid, by Fujifilm (1 mentions)
2 n morpholino ethanesulfonic acid, by Dojindo Laboratories (1 mentions)
3 aminopyrrolidine, by Merck Group (1 mentions)
Trimesic acid, by Fujifilm (1 mentions)
2 protocols using 3 mm zirconia beads
1
Metabolomic Analysis of Fecal Samples
2
Quantitative Metabolite Analysis of Quadriceps
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A quantitative analysis of charged metabolites in the quadriceps femoris was performed using CE-TOFMS as described previously (66 (link), 67 (link)). Quadriceps femoris muscle samples (50 mg) were submerged in methanol (500 μl) containing internal standards [methionine sulfone (Wako, Tokyo, Japan), 2-(N-morpholino)ethane sulfonic acid (Dojindo Laboratories, Tokyo, Japan), and d -camphor-10-sulfonic acid (Wako) each at 20 μM]. The mixture was combined with four 3-mm zirconia beads (BioSpec Products, Bartlesville, OK, USA) and vigorously shaken for 5 min using a Shake Master NEO (Biomedical Science, Tokyo, Japan). Subsequently, deionized water (200 μl) and chloroform (500 μl) were added, and the suspension was vigorously shaken for 5 min and centrifuged at 4600g and 4°C for 30 min. The supernatant was filtered through a 5-kDa-cutoff filter column (Millipore, Burlington, MA, USA). The filtrate was concentrated via centrifugation (3 hours at 40°C) and dissolved in water containing reference compounds [200 μM each of 3-aminopyrrolidine (Sigma-Aldrich, St. Louis, MO, USA) and trimesic acid (Wako)] before CE-TOFMS analysis. As described previously (68 (link)), the CE-TOFMS experiments were performed using an Agilent CE-TOFMS system (Agilent Technologies).
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