Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA241718

Local Sample ID:	1961
Subject ID:	SU002500
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Sample Preparation:

Sampleprep ID:	SP002506
Sampleprep Summary:	Sample preparation was performed using a biphasic extraction64 with water, methanol, and methyl tert-butyl ether to separate polar and non-polar metabolites. Capsule device supernatant and stool samples were prepared separately because device samples were liquid and stool samples were solid. For each supernatant sample, 10 µL were aliquoted into one well of a deep sample preparation 96-well plate in a pre-determined randomized order. Samples were extracted one 96-well plate at a time and all steps were carried out at 4 °C unless otherwise specified. Between every 10 experimental samples, a method blank and external QC sample were prepared. Blanks used 10 µL of LC-MS grade water instead of sample, and QC samples used 10 µL of a pooled sample of human gastrointestinal tract contents from unrelated studies. One hundred seventy microliters of methanol containing SPLASH LIPIDOMIX Mass Spec Standard (Avanti) were added to each well and the plate was heat-sealed with foil, shaken vigorously for 30 s at room temperature, unsealed, and 490 µL of methyl tert-butyl ether (MTBE) were added. The plate was then heat-sealed again, vortexed vigorously for 30 s at room temperature, and shaken for 5 min on an orbital shaker. The foil seal was removed and 150 µL of LC-MS grade water were added to each well. The plate was vortexed for 30 s at room temperature and centrifuged at 2400 rpm for 12 min. The foil was removed from the deep-well plate and two 180-µL aliquots of the top phase were transferred to two 96-well Vanquish LC plate using a 12-channel pipette. Two 50-µL aliquots of the aqueous phase were then transferred to two other 96-well Vanquish LC plates. All 96-well plates were dried completely under vacuum at room temperature, heat sealed with foil, and stored at -80 °C until further analysis. Each stool sample was prepared by mixing with spatula and 5±1 mg were transferred to a 2-mL microcentrifuge tube. Two hundred twenty-five microliters of methanol containing SPLASH LIPIDOMIX Mass Spec Standard (Avanti) were added to all microcentrifuge tubes and the tubes were vortexed for 10 s at room temperature. Seven hundred fifty microliters of MTBE and two 3-mm stainless steel balls were added to each tube and samples were homogenized in a Geno/Grinder (SPEX) at 1500 rpm for 1 min. One hundred eighty-eight microliters of water were added to each tube and each tube was vortexed for 30 s at room temperature. Tubes were centrifuged at 14,000 rcf for 2 min at room temperature. Two aliquots of 180 µL of the organic phase were transferred to two 96-well plates. Two 50-µL aliquots of the aqueous phase were transferred to two 96-well plates. All plates were dried completely in a rotary vacuum evaporator, heat-sealed with foil, and stored at -80 °C until further analysis.

Sampleprep ID:

SP002506

Sampleprep Summary:

Sample preparation was performed using a biphasic extraction64 with water, methanol, and methyl tert-butyl ether to separate polar and non-polar metabolites. Capsule device supernatant and stool samples were prepared separately because device samples were liquid and stool samples were solid. For each supernatant sample, 10 µL were aliquoted into one well of a deep sample preparation 96-well plate in a pre-determined randomized order. Samples were extracted one 96-well plate at a time and all steps were carried out at 4 °C unless otherwise specified. Between every 10 experimental samples, a method blank and external QC sample were prepared. Blanks used 10 µL of LC-MS grade water instead of sample, and QC samples used 10 µL of a pooled sample of human gastrointestinal tract contents from unrelated studies. One hundred seventy microliters of methanol containing SPLASH LIPIDOMIX Mass Spec Standard (Avanti) were added to each well and the plate was heat-sealed with foil, shaken vigorously for 30 s at room temperature, unsealed, and 490 µL of methyl tert-butyl ether (MTBE) were added. The plate was then heat-sealed again, vortexed vigorously for 30 s at room temperature, and shaken for 5 min on an orbital shaker. The foil seal was removed and 150 µL of LC-MS grade water were added to each well. The plate was vortexed for 30 s at room temperature and centrifuged at 2400 rpm for 12 min. The foil was removed from the deep-well plate and two 180-µL aliquots of the top phase were transferred to two 96-well Vanquish LC plate using a 12-channel pipette. Two 50-µL aliquots of the aqueous phase were then transferred to two other 96-well Vanquish LC plates. All 96-well plates were dried completely under vacuum at room temperature, heat sealed with foil, and stored at -80 °C until further analysis. Each stool sample was prepared by mixing with spatula and 5±1 mg were transferred to a 2-mL microcentrifuge tube. Two hundred twenty-five microliters of methanol containing SPLASH LIPIDOMIX Mass Spec Standard (Avanti) were added to all microcentrifuge tubes and the tubes were vortexed for 10 s at room temperature. Seven hundred fifty microliters of MTBE and two 3-mm stainless steel balls were added to each tube and samples were homogenized in a Geno/Grinder (SPEX) at 1500 rpm for 1 min. One hundred eighty-eight microliters of water were added to each tube and each tube was vortexed for 30 s at room temperature. Tubes were centrifuged at 14,000 rcf for 2 min at room temperature. Two aliquots of 180 µL of the organic phase were transferred to two 96-well plates. Two 50-µL aliquots of the aqueous phase were transferred to two 96-well plates. All plates were dried completely in a rotary vacuum evaporator, heat-sealed with foil, and stored at -80 °C until further analysis.