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MB Sample ID: SA195437

Local Sample ID:	BileAcid_LI_022421_Stdm5_05
Subject ID:	SU002156
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

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Subject:

Subject ID:	SU002156
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
BileAcid_LI_022421_Stdm5_05	SA195437	FL023917	Standard	Treatment

Collection:

Collection ID:	CO002149
Collection Summary:	Fifteen healthy subjects were enrolled in this study, and each swallowed at least 17 devices over the course of three days. Capsules were retrieved from stool. Every bowel movement during the study was immediately frozen by the subject at -20 °C.
Sample Type:	Intestine

Treatment:

Treatment ID:	TR002168
Treatment Summary:	Capsule types 1 to 4 were designed to sample the upper, middle, middle, and distal small intestine respectively. Stool samples were analyzed alongside samples from capsules.

Sample Preparation:

Sampleprep ID:	SP002162
Sampleprep Summary:	Supernatants from intestinal samples were extracted using a modified 96-well plate biphasic extraction. 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 consisting 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. Multiple dilutions were prepared for bile acid analysis as follows. The dried samples described above were dissolved in 60 µL of bile acid-run solvent (1:1 acetonitrile/ methanol (v/v) containing 6 isotopically labeled bile acid standards at 100 ng/mL) via 30 s of vortexing and 5 min of shaking on an orbital shaker. From this plate, 5 µL were transferred to a new 96-well plate and combined with 145 µL of bile acid-run solvent. Both dilutions were analyzed for all samples, and samples that still presented bile acids above the highest concentration of the standard curve (1500 ng/mL) were diluted 5:145 once more and re-analyzed. A 9-point standard curve that ranged from 0.2 ng/mL to 1500 ng/mL was used with all samples. The standard curve solutions were created by drying bile acid standard solutions to achieve the desired mass of bile acid standards and then dissolved in bile acid-run solvent. Three standard-curve concentration measurements were analyzed after every 20 samples during data acquisition along with one method blank. Approximately 4 mg (±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:

SP002162

Sampleprep Summary:

Supernatants from intestinal samples were extracted using a modified 96-well plate biphasic extraction. 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 consisting 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. Multiple dilutions were prepared for bile acid analysis as follows. The dried samples described above were dissolved in 60 µL of bile acid-run solvent (1:1 acetonitrile/ methanol (v/v) containing 6 isotopically labeled bile acid standards at 100 ng/mL) via 30 s of vortexing and 5 min of shaking on an orbital shaker. From this plate, 5 µL were transferred to a new 96-well plate and combined with 145 µL of bile acid-run solvent. Both dilutions were analyzed for all samples, and samples that still presented bile acids above the highest concentration of the standard curve (1500 ng/mL) were diluted 5:145 once more and re-analyzed. A 9-point standard curve that ranged from 0.2 ng/mL to 1500 ng/mL was used with all samples. The standard curve solutions were created by drying bile acid standard solutions to achieve the desired mass of bile acid standards and then dissolved in bile acid-run solvent. Three standard-curve concentration measurements were analyzed after every 20 samples during data acquisition along with one method blank. Approximately 4 mg (±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	AN003380
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Thermo Vanquish
Column	Waters Acquity BEH C18 (100 x 2mm,1.7um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Thermo TSQ Vantage
Ion Mode	NEGATIVE
Units	ng/mL

Chromatography:

Chromatography ID:	CH002498
Chromatography Summary:	Samples were analyzed using a Thermofisher Vanquish UHPLC system coupled to a Thermofisher TSQ Altis triple-quadrupole mass spectrometer. An Aquity BEH C18 column (1.7 µm, 2.1 mm×100 mm) with guard column Acquity BEH C18 (1.7 µm, 2.1 mm×5 mm) was used for chromatographic separation with mobile phases of A: LC-MS-grade water with 0.1% formic acid, and B: LC-MS-grade acetonitrile with 0.1% formic acid with a flow rate of 400 µL/min and column temperature of 50 °C. The gradient began at 20% B for 1 min, then shifted to 45% B between 1 and 11 min, then to 95% B between 11 and 14 min, then to 99% B between 14 and 14.5 min, 99% B was maintained until 15.5 min, then transitioned from 99% B to 20% B between 15.5 and 16.5 min, and maintained at 20% B until 18 min. Injection volume was 5 µL and MRM scans were collected for all bile acids and internal standards.
Instrument Name:	Thermo Vanquish
Column Name:	Waters Acquity BEH C18 (100 x 2mm,1.7um)
Column Temperature:	50
Flow Gradient:	The gradient began at 20% B for 1 min, then shifted to 45% B between 1 and 11 min, then to 95% B between 11 and 14 min, then to 99% B between 14 and 14.5 min, 99% B was maintained until 15.5 min, then transitioned from 99% B to 20% B between 15.5 and 16.5 min, and maintained at 20% B until 18 min.
Flow Rate:	400 uL/min
Sample Injection:	5 ul
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003147
Analysis ID:	AN003380
Instrument Name:	Thermo TSQ Vantage
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	MRM scans were imported to Skyline58 software. Skyline performed peak integration for all analytes with given mass transitions and retention time windows (Table S2). The chromatogram for each analyte was manually checked to confirm correct peak integration. Peak area was exported for all analytes. Bile acid chemical structures were removed if there was not a convincing chromatographic peak observed in ≥1 sample. The ratio of analyte to its closest eluting internal standard was calculated and used for quantification. A linear model was fitted to standard curve points for each bile acid (R2>0.995 for all bile acids) and the model was applied to all samples and blanks to calculate concentrations. The average concentration reported for method blanks was subtracted from sample concentrations. Since multiple dilutions were analyzed for each sample, the measurement closest to the center of the standard curve (750 ng/mL) was used. Zero values were imputed with a concentration value between 0.001 and 0.1 ng/mL. Concentrations were reported as ng/mL for intestinal sample liquid supernatant, and ng/g for wet stool.
Ion Mode:	NEGATIVE