2.5. Mass spectrometry (MS) for metabolomics analysis

Metabolomics analysis was performed by Metabolon, Inc. (Durham, NC, USA), as previously described [22]. Both muscle and serum were methanol extracted and analyzed by ultra-high-performance liquid chromatography-tandem MS (UPLC-MS/MS; positive mode), UPLC-MS/MS (negative mode) and gas chromatography–MS (GC–MS). The UPLC-MS/MS platform utilized a Waters Acquity UPLC with Waters UPLC BEH C18-2.1 × 100 mm, 1.7 μm columns and a ThermoFisher LTQ MS, which included an electrospray ionization source and a linear ion-trap mass analyzer. Samples destined for analysis by GC–MS were dried under vacuum desiccation for a minimum of 18 h prior to being derivatized using bis(trimethylsilyl) trifluoroacetamide. Derivatized samples were separated on a 5% phenyldimethyl silicone column with helium as carrier gas and a temperature ramp from 60 °C to 340 °C within a 17-min period. All samples were analyzed on a Thermo-Finnigan Trace DSQ fast-scanning single-quadrupole MS operated at unit mass resolving power with electron impact ionization and a 50–750 atomic mass unit scan range. Metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as associated MS spectra and were curated by visual inspection for quality control using software developed at Metabolon, Inc [23].