Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA254989

Local Sample ID:	AS_29
Subject ID:	SU002635
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

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Sample Preparation:

Sampleprep ID:	SP002641
Sampleprep Summary:	SCFA analysis was performed using an Agilent 6490 series triple quadrupole mass spectrometer (Agilent Technologies) with chromatographic separation on an Agilent 1200 series high-performance liquid chromatography system (HPLC) (Agilent Technologies). SCFAs were extracted by adding 360μL of 50% acetonitrile with 10μM 4-methylvaleric acid internal standard to 40μL of biological sample supernatant. Samples were then vortexed for 30 seconds, incubated at 10oC for 30 minutes at 950RPM, centrifuged at 14,000RPM for five minutes at 4oC, followed by supernatant collection. Derivatisation for SCFA analysis was performed by first adding 20μL of 20μM 13C6-nitrophenylhydrazine as internal standard to 40μL of the extracted supernatant, followed by 20μL each of 200mM nitrophenylhydrazine and 120mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), incubated at 40oC for 30 minutes at 950RPM, quenched with 20μL of 200mM quinic acid, and incubated at 40oC for a further 30 minutes at 950RPM. Lastly, the samples were reconstituted with 1.9mL of 15% acetonitrile and 1μL was injected onto the column. Pooled biological quality controls (PBQCs) were created by pooling extracts (20μL) from individual biological samples and injected onto the column in five sample intervals. A reagent and procedural blank of the original sample preservation buffer was included for analysis to perform background correction. Polar metabolite analysis was performed using an Agilent 6545 series quadrupole time-of-flight mass spectrometer (Agilent Technologies) with chromatographic separation on an Agilent 1200 series HPLC system (Agilent Technologies). Metabolite extraction was performed by first adding a solvent mixture of acetonitrile, methanol and water to 20μL of biological sample, followed by vortexing, sonication and agitation. Samples were then centrifuged and supernatant collected and mixed with an internal standard mixture containing 13C5, 15N-valine, 13C6-leucine, and 13C6-sorbitol, and 14μL of sample was injected onto the column. Samples were injected in a randomised order and PBQCs were injected onto the column in five sample intervals. Data matrices were imported to the web-based platform MetaboAnalyst (v5.0) for quality control checks by multivariate statistics. SCFA data were normalised to internal standards, and polar metabolite data were log-transformed and median-normalised.

Sampleprep ID:

SP002641

Sampleprep Summary:

SCFA analysis was performed using an Agilent 6490 series triple quadrupole mass spectrometer (Agilent Technologies) with chromatographic separation on an Agilent 1200 series high-performance liquid chromatography system (HPLC) (Agilent Technologies). SCFAs were extracted by adding 360μL of 50% acetonitrile with 10μM 4-methylvaleric acid internal standard to 40μL of biological sample supernatant. Samples were then vortexed for 30 seconds, incubated at 10oC for 30 minutes at 950RPM, centrifuged at 14,000RPM for five minutes at 4oC, followed by supernatant collection. Derivatisation for SCFA analysis was performed by first adding 20μL of 20μM 13C6-nitrophenylhydrazine as internal standard to 40μL of the extracted supernatant, followed by 20μL each of 200mM nitrophenylhydrazine and 120mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), incubated at 40oC for 30 minutes at 950RPM, quenched with 20μL of 200mM quinic acid, and incubated at 40oC for a further 30 minutes at 950RPM. Lastly, the samples were reconstituted with 1.9mL of 15% acetonitrile and 1μL was injected onto the column. Pooled biological quality controls (PBQCs) were created by pooling extracts (20μL) from individual biological samples and injected onto the column in five sample intervals. A reagent and procedural blank of the original sample preservation buffer was included for analysis to perform background correction. Polar metabolite analysis was performed using an Agilent 6545 series quadrupole time-of-flight mass spectrometer (Agilent Technologies) with chromatographic separation on an Agilent 1200 series HPLC system (Agilent Technologies). Metabolite extraction was performed by first adding a solvent mixture of acetonitrile, methanol and water to 20μL of biological sample, followed by vortexing, sonication and agitation. Samples were then centrifuged and supernatant collected and mixed with an internal standard mixture containing 13C5, 15N-valine, 13C6-leucine, and 13C6-sorbitol, and 14μL of sample was injected onto the column. Samples were injected in a randomised order and PBQCs were injected onto the column in five sample intervals. Data matrices were imported to the web-based platform MetaboAnalyst (v5.0) for quality control checks by multivariate statistics. SCFA data were normalised to internal standards, and polar metabolite data were log-transformed and median-normalised.