Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA236746

Local Sample ID:	100003157
Subject ID:	SU002444
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Sample Preparation:

Sampleprep ID:	SP002450
Sampleprep Summary:	Stool samples were weighed into bead blaster tubes with zircon beads and homogenized in 100% methanol to a final concentration of 10mg/mL including deuterated SCFA internal standards Chun et al. p.22 (CDN Isotopes). 46 Control samples were created by combining sample homogenates into a 1mL of pooled sample (pooled control) or 1mL of methanol (instrument blank). A portion of the pooled control was then subjected to three successive rounds of extraction to deplete SCFAs but retain the insoluble particles to generate null matrix material. A standard curve for the cocktail of SCFAs was prepared in null matrix, and these samples were extracted side-by-side with the study samples and sequence blanks (either methanol or null matrix). The resulting SCFA extracts were then derivatized as described47 for liquid chromatography tandem mass spectrometry (LCMS) with methanol being used instead of acetonitrile. Samples were analyzed with a QExactive HF coupled to a Dionex Ultimate 3000. A Waters BEH C18 column (2.1 x 150mm, 1.7µm) was used with Buffer A as 0.1% formic acid in water, and Buffer B as 0.1%formic acid in acetonitrile. A gradient elution was used (15%B to 55%B in 9 min, 200µL/min) and the mass spectrometer was operated in negative ion mode (HESI, -3.5kV). The order of acquisition was randomized and several control blocks including blanks and external standards were interspersed throughout the run to assess instrument performance and quality control of the derivatization protocol. All analytes were corrected to their respective internal standard, and the resulting ratio was interpolated against the matrix-controlled standard curve to quantify the level of SCFA in each sample. The limit of detection was determined from null-matrix samples for each analyte, and this limit was imputed for any point falling below the limit of detection. The interpolated values were used for the downstream analyses, and the imputed ratio threshold values were used for samples below the limit of detection.

Sampleprep ID:

SP002450

Sampleprep Summary:

Stool samples were weighed into bead blaster tubes with zircon beads and homogenized in 100% methanol to a final concentration of 10mg/mL including deuterated SCFA internal standards Chun et al. p.22 (CDN Isotopes). 46 Control samples were created by combining sample homogenates into a 1mL of pooled sample (pooled control) or 1mL of methanol (instrument blank). A portion of the pooled control was then subjected to three successive rounds of extraction to deplete SCFAs but retain the insoluble particles to generate null matrix material. A standard curve for the cocktail of SCFAs was prepared in null matrix, and these samples were extracted side-by-side with the study samples and sequence blanks (either methanol or null matrix). The resulting SCFA extracts were then derivatized as described47 for liquid chromatography tandem mass spectrometry (LCMS) with methanol being used instead of acetonitrile. Samples were analyzed with a QExactive HF coupled to a Dionex Ultimate 3000. A Waters BEH C18 column (2.1 x 150mm, 1.7µm) was used with Buffer A as 0.1% formic acid in water, and Buffer B as 0.1%formic acid in acetonitrile. A gradient elution was used (15%B to 55%B in 9 min, 200µL/min) and the mass spectrometer was operated in negative ion mode (HESI, -3.5kV). The order of acquisition was randomized and several control blocks including blanks and external standards were interspersed throughout the run to assess instrument performance and quality control of the derivatization protocol. All analytes were corrected to their respective internal standard, and the resulting ratio was interpolated against the matrix-controlled standard curve to quantify the level of SCFA in each sample. The limit of detection was determined from null-matrix samples for each analyte, and this limit was imputed for any point falling below the limit of detection. The interpolated values were used for the downstream analyses, and the imputed ratio threshold values were used for samples below the limit of detection.