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MB Sample ID: SA254960
Local Sample ID: | AS_28 |
Subject ID: | SU002635 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Male and female |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002635 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Male and female |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
AS_28 | SA254960 | FL032651 | 2 | CI_group |
Collection:
Collection ID: | CO002628 |
Collection Summary: | Stool was collected and stored using Norgen Stool Nucleic Acid Collection and Preservation Tubes (Norgen Biotek, ON, Canada). |
Sample Type: | Feces |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR002647 |
Treatment Summary: | Stool samples containing buffer were vortexed vigorously and 1 ml was transferred to a clean 2 ml tube. Samples were centrifuged for 20 min at 13,000 xg at 4°C and the supernatant was transferred to a clean 2 ml screw-cap tube for storage. |
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. |
Combined analysis:
Analysis ID | AN004170 |
---|---|
Analysis type | MS |
Chromatography type | HILIC |
Chromatography system | Agilent 1200 |
Column | Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um) |
MS Type | ESI |
MS instrument type | QTOF |
MS instrument name | Agilent 6545 QTOF |
Ion Mode | NEGATIVE |
Units | ng/ml |
Chromatography:
Chromatography ID: | CH003088 |
Instrument Name: | Agilent 1200 |
Column Name: | Merck SeQuant ZIC-HILIC (150 x 2.1mm,5um) |
Column Temperature: | 40 |
Flow Gradient: | time = 0 min, 90% B; t = 0.5 min, 90% B; t = 12 min, 40% B; t = 14 min, 40% B; t = 15 min, 5%; t = 18 min, 5% B; and t = 19 min, 90%. |
Flow Rate: | 250 ul/min |
Solvent A: | 100% water; 20 mM ammonium carbonate (pH 9.0) |
Solvent B: | 100% acetonitrile |
Chromatography Type: | HILIC |
MS:
MS ID: | MS003917 |
Analysis ID: | AN004170 |
Instrument Name: | Agilent 6545 QTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | Negative mode LC-MS data were collected in centroid mode with a scan range of 50 to 1700 mass-to-charge ratio (m/z) and an acquisition rate of 1.2 spectra/s. Samples were analyzed in the same analytical batch and randomized with a quality control every five samples. Authentic standards were also run to generate the library for targeted analysis. Level 1 metabolite identification [according to the Metabolite Standard Initiative] was based on matching accurate mass, retention time, and tandem MS (MS/MS) spectra to the 550 authentic standards in the MA in-house library. Metabolite abundance based on area under the curve (AUC) was obtained using Agilent Masshunter Quantitative Analysis B 0.7.00. Statistical analysis was performed applying the web-based platform MetaboAnalyst applying no missing value imputation, normalization to median peak area, and no scaling or transformation. RAW_FILE_NAME=AS_01.mzML to AS_35.mzML refer to metabolite detection of short-chain fatty acids, and RAW_FILE_NAME=AS_001.mzML to AS_035.mzML refer the polar metabolites. |
Ion Mode: | NEGATIVE |