Summary of Study ST000224

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000182. The data can be accessed directly via it's Project DOI: 10.21228/M8M888 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST000224
Study Title	Vitamin targeted metabolomics of the small intestine in malnourished and control mice
Study Type	Targeted metabolomics
Study Summary	A total of 8 samples from 6 week old, female C57BL/6 mice, treated for 3 weeks with a malnourished diet or a control-fed isocaloric diet. Samples were taken from the small intestinal fecal content at the terminus of the ileum for targeted analysis of vitamin concentrations.
Institute	University of Victoria
Department	The Uvic Proteomics and Metabolomics Innovation Centre
Last Name	Borchers
First Name	Christoph
Address	#3101-4464 Markham St Victoria, BC Canada, V8Z 7X8
Email	christoph@proteincentre.com
Submit Date	2015-06-08
Num Groups	2
Total Subjects	8
Raw Data Available	No
Analysis Type Detail	LC-MS
Release Date	2015-06-08
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP000245
Sampleprep Summary:	Vitamin-targeted metabolomics. The samples were analyzed by UPLC-MRM/MS on a Dionex UltiMate 3400 RSLC system coupled to an AB Sciex 4000 QTRAP triple-quadrupole mass spectrometer equipped with an electrospray ionization source. The standard substances of vitamin A (retinal, retinol, retinoic acid), B1 (thiamine), B2 (riboflavin), B3 (niacinamide), B6 (pydidoximine, pyridoxine, pyridoxal, pyridoxal-mono-phosphate), B7 (biotin), B9 (folic acid), D2, D3, E (?-tocopherol, ?-tocopherol, and ?-tocotrienol), K1 and K2, were purchased either from Sigma-Aldrich or from Cayman Chemicals Inc. The MRM transitions of individual analytes were optimized by direct infusion of a standard solution of each compound into the MS instrument. Each sample was added with a methanolic BHT (2 mg/mL) solution at a ratio of 15 ?L per mg of the small intestine digestate. Vitamins were extracted by homogenizing the samples at a shaking frequency of 30 Hz for 1 min twice using a Retsch MM400 mixer mill and with the aid of two 3-mm stainless steel metal balls, followed by 5-min sonication in an icy water bath. The samples were then centrifuged in a micro-centrifuge at 12,500 rpm and 4oC for 10 min. A 300-?L aliquot of the supernatant was transferred into a 3-mL borosilicate glass test tube and mixed with 300 ?L of water and 900 ?L of hexane. After 1 min vortex mixing, the tubes were centrifuged at 4000 rpm and 10 oC in a Beckman R22 centrifuge to separate the supernatant organic phase from the lower aqueous phase. The supernatants were carefully pipetted out to another sets of 3-mL test tubes. The fat-soluble vitamins were further extracted from the aqueous phase with 900 ?L of hexane two more times. After liquid-liquid extraction, the pooled organic phase for each sample was dried down in a speed-vacuum concentrator at room temperature. The dried residue was reconstituted in 100 ?L of ethanol. A 20-?L aliquot was injected for quantitation of the fat-soluble vitamins by LC-(+)ESI-MRM/MS on Waters BEH C18 (2.1 x 50 mm, 1.7 ?m) UPLC column and with 0.1% formic acid in water and acetonitrile as the mobile phase for binary solvent gradient elution. An efficient elution gradient was 50% to 100% B in 10 min. The column temperature was 50oC and the flow rate was 300 ?L/min. The aqueous phases were loaded onto reversed-phase polymeric HLB cartridges (60 mg/1mL, Waters Inc.), which have been activated with 1 mL of methanol and equilibrated with 1 mL of 50% methanol before use. Under a 5-inch Hg vaccum, the flow-throw fractions were collected and the resins were washed with 1 mL of 50% methanol with the flow-through fractions collected. The pooled flow-through fractions were dried in a nitrogen evaporator at 30 oC. The residue from each sample was reconstituted in 100 ?L of 2% methanol. A 20-?L aliquot was injected for quantitation of the water-soluble vitamins by UPLC-MRM/MS with (+) or (-) ESI and on a Waters BEH C18 (2.1 x 150 mm, 1.7 ?m) UPLC column and using 0.01% formic acid in water and methanol as the mobile phase for binary solvent gradient elution. The efficient elution gradient was 2% B for 0.5 min and 2% to 50% B in 8 min. The column temperature was 30 oC and the flow rate was 250 ?L/min. The concentrations of all the detected vitamins were calculated from the standard calibration curves of individual vitamins, which were prepared with the use of their authentic compounds.

Sampleprep ID:

SP000245

Sampleprep Summary:

Vitamin-targeted metabolomics. The samples were analyzed by UPLC-MRM/MS on a Dionex UltiMate 3400 RSLC system coupled to an AB Sciex 4000 QTRAP triple-quadrupole mass spectrometer equipped with an electrospray ionization source. The standard substances of vitamin A (retinal, retinol, retinoic acid), B1 (thiamine), B2 (riboflavin), B3 (niacinamide), B6 (pydidoximine, pyridoxine, pyridoxal, pyridoxal-mono-phosphate), B7 (biotin), B9 (folic acid), D2, D3, E (?-tocopherol, ?-tocopherol, and ?-tocotrienol), K1 and K2, were purchased either from Sigma-Aldrich or from Cayman Chemicals Inc. The MRM transitions of individual analytes were optimized by direct infusion of a standard solution of each compound into the MS instrument. Each sample was added with a methanolic BHT (2 mg/mL) solution at a ratio of 15 ?L per mg of the small intestine digestate. Vitamins were extracted by homogenizing the samples at a shaking frequency of 30 Hz for 1 min twice using a Retsch MM400 mixer mill and with the aid of two 3-mm stainless steel metal balls, followed by 5-min sonication in an icy water bath. The samples were then centrifuged in a micro-centrifuge at 12,500 rpm and 4oC for 10 min. A 300-?L aliquot of the supernatant was transferred into a 3-mL borosilicate glass test tube and mixed with 300 ?L of water and 900 ?L of hexane. After 1 min vortex mixing, the tubes were centrifuged at 4000 rpm and 10 oC in a Beckman R22 centrifuge to separate the supernatant organic phase from the lower aqueous phase. The supernatants were carefully pipetted out to another sets of 3-mL test tubes. The fat-soluble vitamins were further extracted from the aqueous phase with 900 ?L of hexane two more times. After liquid-liquid extraction, the pooled organic phase for each sample was dried down in a speed-vacuum concentrator at room temperature. The dried residue was reconstituted in 100 ?L of ethanol. A 20-?L aliquot was injected for quantitation of the fat-soluble vitamins by LC-(+)ESI-MRM/MS on Waters BEH C18 (2.1 x 50 mm, 1.7 ?m) UPLC column and with 0.1% formic acid in water and acetonitrile as the mobile phase for binary solvent gradient elution. An efficient elution gradient was 50% to 100% B in 10 min. The column temperature was 50oC and the flow rate was 300 ?L/min. The aqueous phases were loaded onto reversed-phase polymeric HLB cartridges (60 mg/1mL, Waters Inc.), which have been activated with 1 mL of methanol and equilibrated with 1 mL of 50% methanol before use. Under a 5-inch Hg vaccum, the flow-throw fractions were collected and the resins were washed with 1 mL of 50% methanol with the flow-through fractions collected. The pooled flow-through fractions were dried in a nitrogen evaporator at 30 oC. The residue from each sample was reconstituted in 100 ?L of 2% methanol. A 20-?L aliquot was injected for quantitation of the water-soluble vitamins by UPLC-MRM/MS with (+) or (-) ESI and on a Waters BEH C18 (2.1 x 150 mm, 1.7 ?m) UPLC column and using 0.01% formic acid in water and methanol as the mobile phase for binary solvent gradient elution. The efficient elution gradient was 2% B for 0.5 min and 2% to 50% B in 8 min. The column temperature was 30 oC and the flow rate was 250 ?L/min. The concentrations of all the detected vitamins were calculated from the standard calibration curves of individual vitamins, which were prepared with the use of their authentic compounds.