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MB Sample ID: SA193793

Local Sample ID:	CarnsTG2
Subject ID:	SU002138
Subject Type:	Mammal
Subject Species:	Mus musculus
Age Or Age Range:	8-12 weeks
Gender:	Male

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Sample Preparation:

Sampleprep ID:	SP002144
Sampleprep Summary:	To extract polar metabolites, 800 µL of methanol was added to 200 µL of homogenized heart sample. Mixture was vortexed for 2 min and then placed on ice for 10 min, followed by another 2 min of vortex mixing, and centrifuged at 15,000 rpm for 20 min at 4 °C. Supernatant was transferred into a glass vial and dried in a SpeedVac evaporator to remove methanol, followed by lyophilization to remove water. The dried metabolite extract was dissolved with 30 µL of 20 mg/mL methoxyamine hydrochloride pyridine solution followed by vigorous vortex mixing for 1 min. Methoxylation was performed by sonicating the sample for 20 min and incubation at 60 °C for 1 h. Derivatization was performed by adding 20 µL of N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) or N-Methyl-N-tert-butyldimethysilyltrifuoroacetamide (MTBSTFA) with 1% trimethylchlorosilane to the glass vial. Samples were incubated for 1 h at 60 °C and the mixture was transferred to a GC vial for analysis. Pooled samples were prepared by mixing 30 µL of derivatized metabolite extract from each sample to monitor the instrumental variations during the course of GC×GC-MS analysis.

Sampleprep ID:

SP002144

Sampleprep Summary:

To extract polar metabolites, 800 µL of methanol was added to 200 µL of homogenized heart sample. Mixture was vortexed for 2 min and then placed on ice for 10 min, followed by another 2 min of vortex mixing, and centrifuged at 15,000 rpm for 20 min at 4 °C. Supernatant was transferred into a glass vial and dried in a SpeedVac evaporator to remove methanol, followed by lyophilization to remove water. The dried metabolite extract was dissolved with 30 µL of 20 mg/mL methoxyamine hydrochloride pyridine solution followed by vigorous vortex mixing for 1 min. Methoxylation was performed by sonicating the sample for 20 min and incubation at 60 °C for 1 h. Derivatization was performed by adding 20 µL of N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) or N-Methyl-N-tert-butyldimethysilyltrifuoroacetamide (MTBSTFA) with 1% trimethylchlorosilane to the glass vial. Samples were incubated for 1 h at 60 °C and the mixture was transferred to a GC vial for analysis. Pooled samples were prepared by mixing 30 µL of derivatized metabolite extract from each sample to monitor the instrumental variations during the course of GC×GC-MS analysis.