Ultrafree mc plhcc 250 pk for metabolome analysis

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).

The metabolome profiles were evaluated by extracting metabolites from frozen plasma and liver samples by adding 400 μL of methanol including the internal standards (20 μM each of methionine sulfone and D-camphor-10-sulfonic acid (CSA)) was added to the 40 μL of samples. Next, this mixture was then mixed with 120 μL of ultrapure water and 400 μL of chloroform before centrifuging at 10,000 × g for 3 min at 4 °C. Thereafter, proteins and lipids were removed by transferring the aqueous layer to a centrifugal filter tube (UltrafreeMC-PLHCC 250/pk for Metabolome Analysis, Human Metabolome Technologies). The filtrate was centrifugally concentrated and dissolved in 20 μL of ultrapure water that contained reference compounds (200 μM each of 3-aminopyrrolidine and trimesic acid) immediately before CE-TOFMS analysis^{80 (link)}. All CE-TOFMS experiments were performed using the Agilent CE capillary electrophoresis system (Agilent Technologies). Annotation tables were produced from the measurement of standard compounds that were aligned with the datasets according to similar value and normalized migration time. Peak areas were then normalized against those of the internal standards methionine sulfone or CSA for cationic and anionic metabolites, respectively. The concentrations of each metabolite were calculated based on their relative peak areas and concentrations of standard compounds.