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MB Sample ID: SA191185

Local Sample ID:	RS-02343753
Subject ID:	SU002112
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Sample Preparation:

Sampleprep ID:	SP002118
Sampleprep Summary:	Different procedures have to be used for different sample matrices; this SOP focuses mainly on bile acids in blood plasma but includes other current protocols 5.1. Isolation of an enriched bile acid fraction from blood plasma or serum • Prepare a plate map showing location of samples in wells. Include wells with method blanks and controls on each plate. • Per well add 50µL plasma sample (excluding Method Blanks, IS Check and Utak Controls) -Method Blanks: Contains Anti-Oxidant Solution, CUDA/PHAU in MeOH:ACN Final Vol 250uL -IS Check: Contains Anti-Oxidant Solution, SSTD, CUDA/PHAU in MeOH:ACN Final Vol 250uL -Utak Controls: Conatins 50uL Utak Serum, Anti-Oxidant Sol, SSTD, CUDA/PAHU in MeOH:ACN Final Vol 250uL ● To each well, add 25 µL Anti-Oxidant Solution ● To each IS Check, Utak Control and Customer Sample, add 25 µL SSTD (Final Volume 100nM) ● To each well, add 25 µL CUDA/PHAU (Final Volume 100nM) ● Add 50:50 MeOH:ACN to each well for a final volume of 250uL.Cap or seal plate with Silicon or Foil Mat and vortex for 1 minute at speed 7. ● Centrifuge plate for 5 minutes so protein precipitates into firm pellet at the bottom of each well ● Pipette roughly 200uL of supernatant into 96 Well PVDF Filter Plate with additional 96 Well Nunc Sample plate ● Centrifuge PVDF Filter plate containing samples into collection plate ● Cover plate with Silicon Nunc Plug mat if plate is being stored at -20C, Cover with Pre-Slit Mat for LC-MS Analysis 5.2. Isolation of an enriched bile acid fraction from tissue, e.g. liver or spinal cord ● Weigh tissue sample into eppendorf tube (amount depending on bile acid content) ● Add 10uL Anti-Oxidant Solution (0.2 mg/ml solution BHT/EDTA in 1:1 methanol/water) use repeater pipetter or syringe repeater assembly. ● Add 10uL SSTD 1000nM Solution, use repeater pipetter or syringe repeater assembly ● Add 500uL Cold MeOH and 2 steel balls (3 mm diameter) and homogenize with GenoGrinder in two 30 sec intervals at 1500 rpm until a fine suspension is achieved. ● Centrifuge at 2C for 3 minutes spinning at 15,000rcf. Transfer methanol supernatant to 1.5 mL eppendorf vial containing 10uL 20% glycerol solution ● Add 500uL Cold MeOH and repeat homogenization with GenoGrinder in two 30 sec intervals at 1500 rpm until a fine suspension is achieved. Centrifuge again and add to the same 1.5mL tube ● Evaporated to dryness, or if a large number of samples has to be processed to a 96-well plate ● Re-suspend pellet in 100uL CUDA/PHAU 100nM MeOH:ACN ● Shake reconstitution for 1 minute at speed of 7 and let stand on wet ice for 15 minutes protected from light ● Shake reconstitution again and filter using PVDF Filter plate or Durapore Spin Filters. ● Transfer filtrate to glass insert in amber vial and cap or 96 well plate with pre-slit silicon mat ● Store at -20°C until LCMS analysis 5.3. Isolation of an enriched bile acid fraction from fecal water ● Label eppendorf tubes; add additional vials for blanks and controls ● Add 10uL Anti-Oxidant Solution using repeater pipetter or syringe repeater assembly ● Add 10uL SSTD using repeater pipetter or syringe repeater assembly ● Thaw out samples of fecal water, vortex briefly to homogenize, keep on ice ● Pipette 10uL of fecal water sample into labeled vials. ● Add 800uL of cold extraction solvent, acetonitrile containing 5% of ammonia ● Vortex samples, then shake for 6 minutes at 4 degree C ● Centrifuge for 3 minutes ● Transfer supernatant to fresh 1.5 mL eppendorf vials and evaporated to dryness, or for a large number of samples, to a 96-well plate and discard centrifugation residues ● Add 100µL CUDA/PHAU IS 100nM to dried sample, cap and vortex 10 sec to dissolve residues ● Set rack of samples on wet ice for 15min ● Filter using PVDF Plate or individual Spin Filters ● Transfer filtrates to glass insert in amber vial and cap or to 96 Well Plate ● Store at -20°C until LCMS analysis

Sampleprep ID:

SP002118

Sampleprep Summary:

Different procedures have to be used for different sample matrices; this SOP focuses mainly on bile acids in blood plasma but includes other current protocols 5.1. Isolation of an enriched bile acid fraction from blood plasma or serum • Prepare a plate map showing location of samples in wells. Include wells with method blanks and controls on each plate. • Per well add 50µL plasma sample (excluding Method Blanks, IS Check and Utak Controls) -Method Blanks: Contains Anti-Oxidant Solution, CUDA/PHAU in MeOH:ACN Final Vol 250uL -IS Check: Contains Anti-Oxidant Solution, SSTD, CUDA/PHAU in MeOH:ACN Final Vol 250uL -Utak Controls: Conatins 50uL Utak Serum, Anti-Oxidant Sol, SSTD, CUDA/PAHU in MeOH:ACN Final Vol 250uL ● To each well, add 25 µL Anti-Oxidant Solution ● To each IS Check, Utak Control and Customer Sample, add 25 µL SSTD (Final Volume 100nM) ● To each well, add 25 µL CUDA/PHAU (Final Volume 100nM) ● Add 50:50 MeOH:ACN to each well for a final volume of 250uL.Cap or seal plate with Silicon or Foil Mat and vortex for 1 minute at speed 7. ● Centrifuge plate for 5 minutes so protein precipitates into firm pellet at the bottom of each well ● Pipette roughly 200uL of supernatant into 96 Well PVDF Filter Plate with additional 96 Well Nunc Sample plate ● Centrifuge PVDF Filter plate containing samples into collection plate ● Cover plate with Silicon Nunc Plug mat if plate is being stored at -20C, Cover with Pre-Slit Mat for LC-MS Analysis 5.2. Isolation of an enriched bile acid fraction from tissue, e.g. liver or spinal cord ● Weigh tissue sample into eppendorf tube (amount depending on bile acid content) ● Add 10uL Anti-Oxidant Solution (0.2 mg/ml solution BHT/EDTA in 1:1 methanol/water) use repeater pipetter or syringe repeater assembly. ● Add 10uL SSTD 1000nM Solution, use repeater pipetter or syringe repeater assembly ● Add 500uL Cold MeOH and 2 steel balls (3 mm diameter) and homogenize with GenoGrinder in two 30 sec intervals at 1500 rpm until a fine suspension is achieved. ● Centrifuge at 2C for 3 minutes spinning at 15,000rcf. Transfer methanol supernatant to 1.5 mL eppendorf vial containing 10uL 20% glycerol solution ● Add 500uL Cold MeOH and repeat homogenization with GenoGrinder in two 30 sec intervals at 1500 rpm until a fine suspension is achieved. Centrifuge again and add to the same 1.5mL tube ● Evaporated to dryness, or if a large number of samples has to be processed to a 96-well plate ● Re-suspend pellet in 100uL CUDA/PHAU 100nM MeOH:ACN ● Shake reconstitution for 1 minute at speed of 7 and let stand on wet ice for 15 minutes protected from light ● Shake reconstitution again and filter using PVDF Filter plate or Durapore Spin Filters. ● Transfer filtrate to glass insert in amber vial and cap or 96 well plate with pre-slit silicon mat ● Store at -20°C until LCMS analysis 5.3. Isolation of an enriched bile acid fraction from fecal water ● Label eppendorf tubes; add additional vials for blanks and controls ● Add 10uL Anti-Oxidant Solution using repeater pipetter or syringe repeater assembly ● Add 10uL SSTD using repeater pipetter or syringe repeater assembly ● Thaw out samples of fecal water, vortex briefly to homogenize, keep on ice ● Pipette 10uL of fecal water sample into labeled vials. ● Add 800uL of cold extraction solvent, acetonitrile containing 5% of ammonia ● Vortex samples, then shake for 6 minutes at 4 degree C ● Centrifuge for 3 minutes ● Transfer supernatant to fresh 1.5 mL eppendorf vials and evaporated to dryness, or for a large number of samples, to a 96-well plate and discard centrifugation residues ● Add 100µL CUDA/PHAU IS 100nM to dried sample, cap and vortex 10 sec to dissolve residues ● Set rack of samples on wet ice for 15min ● Filter using PVDF Plate or individual Spin Filters ● Transfer filtrates to glass insert in amber vial and cap or to 96 Well Plate ● Store at -20°C until LCMS analysis