Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA333553

Local Sample ID:	QC6
Subject ID:	SU003218
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Sample Preparation:

Sampleprep ID:	SP003224
Sampleprep Summary:	For CE-MS analysis 200 µL of frozen plasma extract (MeOH/EtOH 1:1, v/v) were thawed on ice and dried completely using a SpeedVac Concentrator System. The resulting residue was then reconstituted in 100 µL of 0.2 mM MetS dissolved in 0.1 M FA. After vortex-mixing for 1 min, the samples were transferred to a Millipore filter with a 30 kDa protein cutoff and centrifuged at 2,000 x g for 40 min at 4 °C. Finally, the resulting ultrafiltrate was transferred to a CE-MS vial for further analysis. Prior to the analysis vials were centrifuged at 2,000 x g for 10 min at 4 °C to ensure that any possible sediment remained at the bottom of the vial. For GC-MS analysis 200 µL of each plasma extract was thawed on ice to room temperature and 30 µL of the internal standard (IS), palmitic acid-d31 in MeOH (80 mg/mL) was added to it. The mixture was vortexed for 5 min and 200 µL of the solution were transferred to a GC-MS vial. Then, samples were evaporated to dryness using a SpeedVac Concentrator System and maintained at 8 °C in the Gerstel Multiple Purpose Sample (MPS) Preparation Station. An automated two step derivatization process was performed before sample injection using a protocol adjusted from a previous reported method. First, each precipitate was redissolved in 20 µL of O-methoxyamine solution (15 mg/mL in pyridine) for the methoximation process, mixed 10 min at 1,000 rpm and incubated for 90 min at 60 °C at 750 rpm. Second, and after waiting 5 min, 40 µL of BSTFA with 1% TMCS were added for the silylation process. Then, samples were mixed for 10 min at 1,000 rpm and incubated for 60 min at 60 °C at 750 rpm. After waiting 30 min at 8 °C, 80 µL of heptane containing 20 mg/mL of tricosane (IS) were added and mixed for 5 min at 1,000 rpm. Finally, samples were maintained at 8 °C for 30 min before injection. For LC-MS 200 µL of each plasma extract was thawed on ice to room temperature and centrifuged for 10 min at 16,100 x g at 4 °C, transferred to a LC-MS vial and directly injected into the system.

Sampleprep ID:

SP003224

Sampleprep Summary:

For CE-MS analysis 200 µL of frozen plasma extract (MeOH/EtOH 1:1, v/v) were thawed on ice and dried completely using a SpeedVac Concentrator System. The resulting residue was then reconstituted in 100 µL of 0.2 mM MetS dissolved in 0.1 M FA. After vortex-mixing for 1 min, the samples were transferred to a Millipore filter with a 30 kDa protein cutoff and centrifuged at 2,000 x g for 40 min at 4 °C. Finally, the resulting ultrafiltrate was transferred to a CE-MS vial for further analysis. Prior to the analysis vials were centrifuged at 2,000 x g for 10 min at 4 °C to ensure that any possible sediment remained at the bottom of the vial. For GC-MS analysis 200 µL of each plasma extract was thawed on ice to room temperature and 30 µL of the internal standard (IS), palmitic acid-d31 in MeOH (80 mg/mL) was added to it. The mixture was vortexed for 5 min and 200 µL of the solution were transferred to a GC-MS vial. Then, samples were evaporated to dryness using a SpeedVac Concentrator System and maintained at 8 °C in the Gerstel Multiple Purpose Sample (MPS) Preparation Station. An automated two step derivatization process was performed before sample injection using a protocol adjusted from a previous reported method. First, each precipitate was redissolved in 20 µL of O-methoxyamine solution (15 mg/mL in pyridine) for the methoximation process, mixed 10 min at 1,000 rpm and incubated for 90 min at 60 °C at 750 rpm. Second, and after waiting 5 min, 40 µL of BSTFA with 1% TMCS were added for the silylation process. Then, samples were mixed for 10 min at 1,000 rpm and incubated for 60 min at 60 °C at 750 rpm. After waiting 30 min at 8 °C, 80 µL of heptane containing 20 mg/mL of tricosane (IS) were added and mixed for 5 min at 1,000 rpm. Finally, samples were maintained at 8 °C for 30 min before injection. For LC-MS 200 µL of each plasma extract was thawed on ice to room temperature and centrifuged for 10 min at 16,100 x g at 4 °C, transferred to a LC-MS vial and directly injected into the system.