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MB Sample ID: SA195489

Local Sample ID:	BileAcid_HI_022421_Stdm9_06
Subject ID:	SU002156
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male and female

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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.