Summary of Study ST003032

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 PR001885. The data can be accessed directly via it's Project DOI: 10.21228/M82F00 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST003032
Study TitleEffects of Preanalytical Sample Collection and Handling on Comprehensive Metabolite Measurements in Human Urine Biospecimens
Study TypeLCMS Investigation of Urine Metabolite Stability
Study SummaryThis study evaluates the impact of sample handling conditions on urine metabolome profiles relative to a gold standard condition. The sample handling conditions included a combination of three variables, use of preservative (None, Borate, or Chlorhexidine), refrigeration storage time (0h or 24h), or freeze-thaws (either 1x or 3x freeze-thaws, thawed on ice, in a refrigerator, or at room temperature). Samples were taken from 13 participants, and then aliquoted and subjected to the various conditions. Comparisons between treatment effects were taken between pairs of samples within each participant’s sample set, a within participant paired design.
Institute
National Center for Advancing Translational Sciences
DepartmentDivision of Pre-clinical Innovation
LaboratoryInformatics Core
Last NameBraisted
First NameJohn
Address9800 Medical Center Drive
Emailjohn.braisted@nih.gov
Phone3014028798
Submit Date2024-01-02
Total Subjects13
Num Males6
Num Females7
Analysis Type DetailOther
Release Date2025-01-02
Release Version1
John Braisted John Braisted
https://dx.doi.org/10.21228/M82F00
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001885
Project DOI:doi: 10.21228/M82F00
Project Title:Effects of Preanalytical Sample Collection and Handling on Comprehensive Metabolite Measurements in Human Urine Biospecimens
Project Type:LCMS
Project Summary:This project evaluates the impact of sample handling conditions on urine metabolome profiles relative to a gold standard condition. The sample handling conditions included a combination of three variables, use of preservative (None, Borate, or Chlorhexidine), refrigeration storage time (0h or 24h), or freeze-thaws (either 1x or 3x freeze-thaws, thawed on ice, in a refrigerator, or at room temperature). Samples were taken from 13 participants, and then aliquoted and subjected to the various conditions. Comparisons between treatment effects were taken between pairs of samples within each participant’s sample set, a within participant paired design.
Institute:National Center for Advancing Tranlastional Sciences
Department:Division of Pre-clinical Innovation
Laboratory:Informatics Core
Last Name:Braisted
First Name:John
Address:9800 Medical Center Drive
Email:john.braisted@nih.gov
Phone:3014028798
Funding Source:ZIC TR000410-05, NCATS/NIH

Subject:

Subject ID:SU003146
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Age Or Age Range:23-64
Weight Or Weight Range:54.4-90.7 kg
Height Or Height Range:158-184 cm
Gender:Male and female
Human Smoking Status:12 - Non-smokers, 1 - Smoker
Species Group:Mammals

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id preservative fridge_store_time thaw
SA3282185607_U26Borate 0 1 fridge
SA3282195611_U26Borate 0 1 fridge
SA3282205601_U26Borate 0 1 fridge
SA3282215609_U26Borate 0 1 fridge
SA3282225605_U26Borate 0 1 fridge
SA3282235612_U26Borate 0 1 fridge
SA3282245604_U26Borate 0 1 fridge
SA3282255610_U26Borate 0 1 fridge
SA3282265602_U26Borate 0 1 fridge
SA3282275603_U26Borate 0 1 fridge
SA3282285606_U26Borate 0 1 fridge
SA3282295608_U26Borate 0 1 fridge
SA3282305613_U26Borate 0 1 fridge
SA3282315602_U25Borate 0 1 ice
SA3282325608_U25Borate 0 1 ice
SA3282335612_U25Borate 0 1 ice
SA3282345610_U25Borate 0 1 ice
SA3282355605_U25Borate 0 1 ice
SA3282365611_U25Borate 0 1 ice
SA3282375613_U25Borate 0 1 ice
SA3282385604_U25Borate 0 1 ice
SA3282395606_U25Borate 0 1 ice
SA3282405603_U25Borate 0 1 ice
SA3282415607_U25Borate 0 1 ice
SA3282425601_U25Borate 0 1 ice
SA3282435609_U25Borate 0 1 ice
SA3282055606_U27Borate 0 1 RT
SA3282065610_U27Borate 0 1 RT
SA3282075609_U27Borate 0 1 RT
SA3282085602_U27Borate 0 1 RT
SA3282095601_U27Borate 0 1 RT
SA3282105611_U27Borate 0 1 RT
SA3282115605_U27Borate 0 1 RT
SA3282125612_U27Borate 0 1 RT
SA3282135607_U27Borate 0 1 RT
SA3282145603_U27Borate 0 1 RT
SA3282155608_U27Borate 0 1 RT
SA3282165613_U27Borate 0 1 RT
SA3282175604_U27Borate 0 1 RT
SA3282575611_U30Borate 0 4 fridge
SA3282585611_U29Borate 0 4 fridge
SA3282595601_U30Borate 0 4 fridge
SA3282605609_U30Borate 0 4 fridge
SA3282615603_U29Borate 0 4 fridge
SA3282625607_U30Borate 0 4 fridge
SA3282635613_U29Borate 0 4 fridge
SA3282645601_U29Borate 0 4 fridge
SA3282655606_U29Borate 0 4 fridge
SA3282665606_U30Borate 0 4 fridge
SA3282675608_U30Borate 0 4 fridge
SA3282685608_U29Borate 0 4 fridge
SA3282695604_U29Borate 0 4 fridge
SA3282705604_U30Borate 0 4 fridge
SA3282715603_U30Borate 0 4 fridge
SA3282725613_U30Borate 0 4 fridge
SA3282735610_U29Borate 0 4 fridge
SA3282745607_U29Borate 0 4 fridge
SA3282755609_U29Borate 0 4 fridge
SA3282765612_U30Borate 0 4 fridge
SA3282775610_U30Borate 0 4 fridge
SA3282785612_U29Borate 0 4 fridge
SA3282795602_U30Borate 0 4 fridge
SA3282805602_U29Borate 0 4 fridge
SA3282815605_U29Borate 0 4 fridge
SA3282825605_U30Borate 0 4 fridge
SA3282835607_U28Borate 0 4 ice
SA3282845606_U28Borate 0 4 ice
SA3282855610_U28Borate 0 4 ice
SA3282865612_U28Borate 0 4 ice
SA3282875603_U28Borate 0 4 ice
SA3282885605_U28Borate 0 4 ice
SA3282895613_U28Borate 0 4 ice
SA3282905601_U28Borate 0 4 ice
SA3282915609_U28Borate 0 4 ice
SA3282925602_U28Borate 0 4 ice
SA3282935608_U28Borate 0 4 ice
SA3282945611_U28Borate 0 4 ice
SA3282955604_U28Borate 0 4 ice
SA3282445611_U31Borate 0 4 RT
SA3282455610_U31Borate 0 4 RT
SA3282465604_U31Borate 0 4 RT
SA3282475605_U31Borate 0 4 RT
SA3282485602_U31Borate 0 4 RT
SA3282495603_U31Borate 0 4 RT
SA3282505609_U31Borate 0 4 RT
SA3282515613_U31Borate 0 4 RT
SA3282525608_U31Borate 0 4 RT
SA3282535601_U31Borate 0 4 RT
SA3282545606_U31Borate 0 4 RT
SA3282555612_U31Borate 0 4 RT
SA3282565607_U31Borate 0 4 RT
SA3282965604_U24Borate 0 none
SA3282975604_U22Borate 0 none
SA3282985604_U23Borate 0 none
SA3282995605_U22Borate 0 none
SA3283005608_U24Borate 0 none
SA3283015605_U24Borate 0 none
SA3283025608_U23Borate 0 none
SA3283035608_U22Borate 0 none
SA3283045609_U23Borate 0 none
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Collection:

Collection ID:CO003139
Collection Summary:Mid-stream spot urine collections were obtained for each participant in the mornings into a sterile urine collection cup with a vacutainer port. Participants were not asked to fast. Aliquots were divided into three separate tubes corresponding to the following preservative treatments: 1) no preservative, 2) borate preservative (BD Vacutainer C&S Urine Tubes, borate 2.63 mg/mL, sodium formate 1.65 mg/mL), 3) chlorhexidine preservative (BD Vacutainer Urinalysis Plus, chlorhexidine 0.4%, sodium propionate 94%, ethyl paraben 5.6%). Tubes were centrifuged at 600xg for 5 min. One mL of urine, with no preservative, from each participant was pooled and mixed to be used as a QC sample, which was run repeatedly with other experimentally treated samples during metabolomics profiling (see section Laboratory assays). Ten aliquots of each of the 3 preservative samples (30 samples total per participant) were transferred into 2mL cryotubes to be subjected to various sample handling conditions, namely refrigeration and free-thaw conditions. Conditions tested included 3 preservative conditions (no preservative, chlorhexidine, and borate), 2 refrigeration conditions (24-hour refrigeration, no refrigeration/snap freezing), and 7 freeze-thaw conditions (no freeze-thaw, thaw on ice once or 4 times, thaw in refrigerator once or four times, and thaw at room temperature once or 4 times). One sample of each preservative treatment was placed in the refrigerator for 24 h before freezing in liquid nitrogen and stored at -80 °C. Remaining samples were flash frozen in liquid nitrogen and stored at -80 °C. They were then subject to various thawing conditions and freeze-thaw cycles. Thawing was performed at either room temperature, in a refrigerator, or on wet ice. Freeze thaws consisted of either a single thaw or 4 consecutive freeze-thaw cycles. Samples undergoing freeze thaws were snap frozen in liquid nitrogen following their thaw. Samples undergoing freeze-thaw cycles had the thaw time standardized based on previous work (previous serum study). Thaws on ice were fixed at 50 min, room temperature thaws were fixed at 10 min, and refrigerator thaws were fixed at 16 h to simulate a 5 PM to 9 AM thaw often used in laboratories.
Sample Type:Urine
Collection Method:Spot collection, into sterile urine collection cup
Collection Frequency:Single Collection
Storage Conditions:Described in summary
Storage Vials:Final Storage in 2ml cryotubes.
Additives:None, Chlorhexidine, or Borate (experimental factors)

Treatment:

Treatment ID:TR003155
Treatment Summary:Conditions tested included 3 preservative conditions (no preservative, chlorhexidine, and borate), 2 refrigeration conditions (24-hour refrigeration, no refrigeration/snap freezing), and 7 freeze-thaw conditions (no freeze-thaw, thaw on ice once or 4 times, thaw in refrigerator once or four times, and thaw at room temperature once or 4 times). 28 samples per participant were treated to a combination of these conditions to evaluate the impact of these various handling conditions on measured metabolite levels. Note that the time for freeze thaws were fixed at 10 minutes for room temperature thaws, 50 minutes for ice thaws, and 16h for refrigerator thaws, to simulate an overnight refrigerator thaw. The study design table indicates to which conditions that each sample was exposed.

Sample Preparation:

Sampleprep ID:SP003152
Sampleprep Summary:After separating into separate tubes, the samples were centrifuged at 600xg for 5 minutes and the supernatent separated into cryovials and underwent various treatments. Snap-frozen samples were sent to Metabolon Inc. for sample extraction and LCMS according to Metabolon established protocols.
Processing Storage Conditions:Described in summary
Extract Storage:Described in summary

Combined analysis:

Analysis ID AN004969 AN004970 AN004971 AN004972
Analysis type MS MS MS MS
Chromatography type Reversed phase Reversed phase Reversed phase HILIC
Chromatography system Waters Acquity Waters Acquity Waters Acquity Waters Acquity
Column Waters Acquity BEH C18 (100 x 2mm, 1.7um) Waters Acquity BEH C18 (100 x 2mm, 1.7um) Waters Acquity BEH C18 (100 x 2mm, 1.7um) Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)
MS Type ESI ESI ESI ESI
MS instrument type Orbitrap Orbitrap Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE POSITIVE NEGATIVE NEGATIVE
Units Peak Area Peak Area Peak Area Peak Area

Chromatography:

Chromatography ID:CH003751
Chromatography Summary:Low pH polar (LC/MS Pos early)
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH C18 (100 x 2mm, 1.7um)
Column Temperature:40-50
Flow Gradient:Linear gradient from 5% B to 80% B over 3.35 minutes
Flow Rate:0.35 mL/min
Solvent A:100% water; 0.1% formic acid; 0.05% PFPA, pH ~2.5
Solvent B:100% methanol; 0.1% formic acid; 0.05% PFPA, pH ~2.5
Chromatography Type:Reversed phase
  
Chromatography ID:CH003752
Chromatography Summary:Low pH Lipophilic (LC/MS Pos late)
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH C18 (100 x 2mm, 1.7um)
Column Temperature:40-50
Flow Gradient:Linear gradient from 40% B to 99.5% B over 1.0 minute, hold 99.5% B for 2.4 minutes.
Flow Rate:0.60 mL/min
Solvent A:100% water; 0.1% formic acid; 0.05% PFPA, pH ~2.5
Solvent B:50% methanol/50% acetonitrile; 0.1% formic acid; 0.05% PFPA, pH ~2.5
Chromatography Type:Reversed phase
  
Chromatography ID:CH003753
Chromatography Summary:High pH (LC/MS Neg)
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH C18 (100 x 2mm, 1.7um)
Column Temperature:40-50
Flow Gradient:Linear gradient from 0.5 to 70% B over 4.0 minutes, then rapid gradient to 99% B in 0.5 minutes.
Flow Rate:0.35 mL/min
Solvent A:100% water; 6.5 mM ammonium bicarbonate, pH 8
Solvent B:95% methanol/5% water; 6.5 mM ammonium bicarbonate
Chromatography Type:Reversed phase
  
Chromatography ID:CH003754
Chromatography Summary:HILIC (LC/MS Polar Neg)
Instrument Name:Waters Acquity
Column Name:Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)
Column Temperature:40-50
Flow Gradient:Linear gradient from 5% B to 50% B in 3.5 minutes, then linear gradient from 50% B to 95% B in 2 minutes.
Flow Rate:0.50 mL/min
Solvent A:15% water/5% methanol/80% acetonitrile; 10 mM ammonium formate, (effective pH 10.16 with NH4OH)
Solvent B:50% water/50% acetonitrile; 10 mM ammonium formate, (effective pH 10.60 with NH4OH)
Chromatography Type:HILIC

MS:

MS ID:MS004709
Analysis ID:AN004969
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Metabolon (LC/MS Pos early)
Ion Mode:POSITIVE
  
MS ID:MS004710
Analysis ID:AN004970
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Metabolon (LC/MS Pos late)
Ion Mode:POSITIVE
  
MS ID:MS004711
Analysis ID:AN004971
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Metabolon (LC/MS Neg)
Ion Mode:NEGATIVE
  
MS ID:MS004712
Analysis ID:AN004972
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Metabolon (LC/MS Polar)
Ion Mode:NEGATIVE
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