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MB Sample ID: SA195962

Local Sample ID:	1479
Subject ID:	SU002158
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

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Subject:

Subject ID:	SU002158
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
1479	SA195962	FL023924	Capsule Type 1	Treatment

Collection:

Collection ID:	CO002151
Collection Summary:	Fifteen healthy subjects were enrolled in this study, and each swallowed 17 devices over the course of three days. All 255 ingested devices were recovered, and no adverse events were reported during the study. Of the 255 ingested devices, 17 contained mainly gas. Ten of the 238 ingested devices that did not contain large amounts of gas provided samples with >0 ng of DNA . Saliva samples were collected after evening meals and immediately frozen. Every bowel movement during the study was immediately frozen by the subject at -20 °C. Subject 1 provided additional samples for assessment of replicability and blooming.
Sample Type:	Intestine

Treatment:

Treatment ID:	TR002170
Treatment Summary:	The capsule sampling device (CapScan®, Envivo Bio Inc, San Carlos CA) consists of a one-way valve capping a hollow elastic bladder. The device is prepared for packaging by evacuating the elastic bladder, folding it in half, and packaging the folded device inside a dissolvable capsule measuring 6.5 mm in diameter×23 mm long, onto which an enteric coating is applied. The target pH was pH 6 for type 1 and type 2 capsules, and pH 7.5 for type 3 and type 4, with type 2 and type 4 having a thicker coating to result in delayed opening. The elastic bladder then unfolds and expands into a tube 6 mm in diameter and 33 mm long, thereby drawing in ~300 µL of gut luminal contents through the one-way valve. The one-way valve maintains the integrity of the sample collected inside the bladder as the device moves through the colon and is exposed to stool.

Treatment ID:

TR002170

Treatment Summary:

The capsule sampling device (CapScan®, Envivo Bio Inc, San Carlos CA) consists of a one-way valve capping a hollow elastic bladder. The device is prepared for packaging by evacuating the elastic bladder, folding it in half, and packaging the folded device inside a dissolvable capsule measuring 6.5 mm in diameter×23 mm long, onto which an enteric coating is applied. The target pH was pH 6 for type 1 and type 2 capsules, and pH 7.5 for type 3 and type 4, with type 2 and type 4 having a thicker coating to result in delayed opening. The elastic bladder then unfolds and expands into a tube 6 mm in diameter and 33 mm long, thereby drawing in ~300 µL of gut luminal contents through the one-way valve. The one-way valve maintains the integrity of the sample collected inside the bladder as the device moves through the colon and is exposed to stool.

Sample Preparation:

Sampleprep ID:	SP002164
Sampleprep Summary:	Supernatants from intestinal samples were extracted using a modified 96-well plate biphasic extraction63. Samples in microcentrifuge tubes were thawed on ice and 10 µL were transferred to wells of a 2-mL polypropylene 96-well plate in a predetermined randomized order. A quality control (QC) sample consisteing of a pool of many intestinal samples from pilot studies was used to assess analytical variation. QC sample matrix (10 µL) and blanks (10 µL of LC-MS grade water) were included for every 10th sample. One hundred seventy microliters of methanol containing UltimateSPLASH Avanti Polar Lipids (Alabaster, Alabama) as an internal standard were added to each well. Then 490 µL of methyl-tert-butyl-ether (MTBE) containing internal standard Cholesterol Ester 22:1 were added to each well. Plates were sealed, vortexed vigorously for 30 s, and shaken on an orbital shaking plate for 5 min at 4 °C. The plate was unsealed and 150 µL of cold water were added to each well. Plates were re-sealed, vortexed vigorously for 30 s, and centrifuged for 12 min at 4000 rcf and 4 °C. From the top phase of the extraction wells, two aliquots of 180 µL each were transferred to new 96-well plates, and two aliquots of 70 µL each from the bottom phase were transferred to two other new 96-well plates. Plates were spun in a rotary vacuum until dry, sealed, and stored at -80 °C until LC-MS/MS analysis. One of the 96-well plates containing the aqueous phase of extract was dissolved in 35 µL of HILIC-run solvent (8:2 acetonitrile/ water, v/v). Five microliters were analyzed using non-targeted HILIC LC-MS/MS analysis. Immediately after HILIC analysis, the 96-well plates were spun in a rotary vacuum until dry, sealed, and stored at -80 °C until targeted bile acid analysis. Approximately 4±1 mg of wet stool were transferred to 2-mL microcentrifuge tubes. Twenty microliters of QC mix were added to microcentrifuge tubes for QC samples. Blank samples were generated using 20 µL of LC-MS grade water. To each tube, 225 µL of ice-cold methanol containing internal standards (as above) were added, followed by 750 µL of ice-cold MTBE with CE 22:1. Two 3-mm stainless-steel grinding beads were added to each tube and tubes were processed in a Geno/Grinder automated tissue homogenizer and cell lyser at 1500 rpm for 1 min. One hundred eighty-eight microliters of cold water were then added to each tube. Tubes were vortexed vigorously and centrifuged at 14,000 rcf for 2 min. Two aliquots of 180 µL each of the MTBE layer and two aliquots of 50 µL each of the lower layer were transferred to four 96-well plates, spun in a rotary vacuum until dry, sealed, and stored at -80 °C until analysis with the intestinal samples. Stool samples were analyzed using HILIC non-targeted LC-MS/MS and diluted in an identical manner to intestinal samples as described above. Stool samples were analyzed in a randomized order after intestinal samples.

Sampleprep ID:

SP002164

Sampleprep Summary:

Supernatants from intestinal samples were extracted using a modified 96-well plate biphasic extraction63. Samples in microcentrifuge tubes were thawed on ice and 10 µL were transferred to wells of a 2-mL polypropylene 96-well plate in a predetermined randomized order. A quality control (QC) sample consisteing of a pool of many intestinal samples from pilot studies was used to assess analytical variation. QC sample matrix (10 µL) and blanks (10 µL of LC-MS grade water) were included for every 10th sample. One hundred seventy microliters of methanol containing UltimateSPLASH Avanti Polar Lipids (Alabaster, Alabama) as an internal standard were added to each well. Then 490 µL of methyl-tert-butyl-ether (MTBE) containing internal standard Cholesterol Ester 22:1 were added to each well. Plates were sealed, vortexed vigorously for 30 s, and shaken on an orbital shaking plate for 5 min at 4 °C. The plate was unsealed and 150 µL of cold water were added to each well. Plates were re-sealed, vortexed vigorously for 30 s, and centrifuged for 12 min at 4000 rcf and 4 °C. From the top phase of the extraction wells, two aliquots of 180 µL each were transferred to new 96-well plates, and two aliquots of 70 µL each from the bottom phase were transferred to two other new 96-well plates. Plates were spun in a rotary vacuum until dry, sealed, and stored at -80 °C until LC-MS/MS analysis. One of the 96-well plates containing the aqueous phase of extract was dissolved in 35 µL of HILIC-run solvent (8:2 acetonitrile/ water, v/v). Five microliters were analyzed using non-targeted HILIC LC-MS/MS analysis. Immediately after HILIC analysis, the 96-well plates were spun in a rotary vacuum until dry, sealed, and stored at -80 °C until targeted bile acid analysis. Approximately 4±1 mg of wet stool were transferred to 2-mL microcentrifuge tubes. Twenty microliters of QC mix were added to microcentrifuge tubes for QC samples. Blank samples were generated using 20 µL of LC-MS grade water. To each tube, 225 µL of ice-cold methanol containing internal standards (as above) were added, followed by 750 µL of ice-cold MTBE with CE 22:1. Two 3-mm stainless-steel grinding beads were added to each tube and tubes were processed in a Geno/Grinder automated tissue homogenizer and cell lyser at 1500 rpm for 1 min. One hundred eighty-eight microliters of cold water were then added to each tube. Tubes were vortexed vigorously and centrifuged at 14,000 rcf for 2 min. Two aliquots of 180 µL each of the MTBE layer and two aliquots of 50 µL each of the lower layer were transferred to four 96-well plates, spun in a rotary vacuum until dry, sealed, and stored at -80 °C until analysis with the intestinal samples. Stool samples were analyzed using HILIC non-targeted LC-MS/MS and diluted in an identical manner to intestinal samples as described above. Stool samples were analyzed in a randomized order after intestinal samples.

Combined analysis:

Analysis ID	AN003382
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Vanquish
Column	Waters Acquity UPLC BEH amide,(150 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	POSITIVE
Units	peak height

Chromatography:

Chromatography ID:	CH002500
Chromatography Summary:	Data acquisition : 3 µL sample aliquots were injected on a Waters Acquity UPLC BEH Amide column (150 mm length × 2.1 mm id; 1.7 μm particle size) maintained at 45°C. A Waters Acquity VanGuard BEH Amide pre-column (5 mm × 2.1 mm id; 1.7 μm particle size) was used as guard column. Mobile phase A was 100% LC-MS grade water with 10 mM ammonium formate and 0.125% formic acid and mobile phase B was 95:5 v/v acetonitrile:water with 10 mM ammonium formate and 0.125% formic acid. Gradient was started at 100% (B) for 2 min, 70% (B) at 7.7 min, 40% (B) at 9.5 min, 30% (B) at 10.25 min, 100% (B) at 12.75 min and isocratic until 16.75 min. The column flow was 0.4 mL/min. Vanquish UHPLC system (ThermoFisher Scientific) was used.
Instrument Name:	Thermo Vanquish
Column Name:	Waters Acquity UPLC BEH amide,(150 x 2.1mm,1.7um)
Flow Gradient:	Gradient was started at 100% (B) for 2 min, 70% (B) at 7.7 min, 40% (B) at 9.5 min, 30% (B) at 10.25 min, 100% (B) at 12.75 min and isocratic until 16.75 min.
Flow Rate:	0.4 mL/min
Solvent A:	100% water; 0.125% formic acid; 10 mM ammonium formate
Solvent B:	95% acetonitrile/5% water 0.125% formic acid; 10 mM ammonium formate
Chromatography Type:	HILIC

MS:

MS ID:	MS003149
Analysis ID:	AN003382
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	A Thermo Q-Exactive HF Orbitrap MS instrument was operated in positive and negative ESI mdoes respectively with the following parameters: mass range 60−900 m/z; spray voltage 3.6kV (ESI+) and −3kV (ESI−), sheath gas (nitrogen) flow rate 60 units; auxiliary gas (nitrogen) flow rate 25 units, capillary temperature 320 ◦C, full scan MS1 mass resolving power 60,000, data-dependent MSMS (dd-MSMS) 4 scans per cycle, normalized collision energy at 20%, 30%, and 40%, dd-MSMS mass resolving power 15,000. Thermo Xcalibur 4.0.27.19 was used for data acquisition and analysis. Instruments was tuned and calibrated by manufacturer’s recommendations. MSDIAL was used for data processing. Conjugated bile acid peak height was converted to relative concentration in ng/mL by comparing to targeted bile acid dataset ST002073.
Ion Mode:	POSITIVE