Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA227360

Local Sample ID:	HCO13
Subject ID:	SU002406
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	5-77
Weight Or Weight Range:	NA
Height Or Height Range:	NA
Gender:	Male and female
Human Lifestyle Factors:	Urban industrial; Rural industrial; Rural traditional; Isolated traditional
Human Alcohol Drug Use:	NA
Human Nutrition:	NA
Human Inclusion Criteria:	NA
Human Exclusion Criteria:	NA

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Treatment:

Treatment ID:	TR002418
Treatment Summary:	The sample preparation protocol used for this project was adapted from a global metabolite extraction protocol with proven success (Want et al. 2013). Samples were thawed and 500 μl of chilled LC-MS grade water (Fisher Scientific) was added to 50 mg of fecal material. Next, a TissueLyzer homogenized samples at 25 Hz for three minutes. Following homogenization, chilled LC-grade methanol (Fisher Scientific) spiked with 4 μM sulfachloropyridazine as the internal standard (IS) was added, bringing the total concentration to 50% methanol. The TissueLyzer homogenized samples again at 25 Hz for three minutes, followed by overnight incubation at 4 °C. The next day, samples were centrifuged at 16,000 x g at 4 °C for ten minutes. Aqueous supernatant was then removed and dried using a SpeedVac vacuum concentrator. Dried extracts were frozen at -80 °C until the day of MS analysis. Immediately prior to MS analysis, extracts were resuspended in 150 μl chilled LC-MS methanol:water (1:1) spiked with 1 μg/ml sulfadimethoxine as a second IS. After resuspension, samples were diluted to a 1:10 ratio. Diluted samples were sonicated using a Fisher Scientific Ultrasonic Cleaning Bath at maximum power for ten minutes. Supernatants were spun briefly to remove any particulates, then loaded into a 96-well plate for MS analysis. One well contained only 150 μl of the resuspension solution to serve as blank control.

Treatment ID:

TR002418

Treatment Summary:

The sample preparation protocol used for this project was adapted from a global metabolite extraction protocol with proven success (Want et al. 2013). Samples were thawed and 500 μl of chilled LC-MS grade water (Fisher Scientific) was added to 50 mg of fecal material. Next, a TissueLyzer homogenized samples at 25 Hz for three minutes. Following homogenization, chilled LC-grade methanol (Fisher Scientific) spiked with 4 μM sulfachloropyridazine as the internal standard (IS) was added, bringing the total concentration to 50% methanol. The TissueLyzer homogenized samples again at 25 Hz for three minutes, followed by overnight incubation at 4 °C. The next day, samples were centrifuged at 16,000 x g at 4 °C for ten minutes. Aqueous supernatant was then removed and dried using a SpeedVac vacuum concentrator. Dried extracts were frozen at -80 °C until the day of MS analysis. Immediately prior to MS analysis, extracts were resuspended in 150 μl chilled LC-MS methanol:water (1:1) spiked with 1 μg/ml sulfadimethoxine as a second IS. After resuspension, samples were diluted to a 1:10 ratio. Diluted samples were sonicated using a Fisher Scientific Ultrasonic Cleaning Bath at maximum power for ten minutes. Supernatants were spun briefly to remove any particulates, then loaded into a 96-well plate for MS analysis. One well contained only 150 μl of the resuspension solution to serve as blank control.