Plasma samples were thawed and analyzed by LC-MS at Emory University as previously described.8 (link)–10 (link, link),15 ,16 (link) Samples were randomized into batches of 20 prior to analysis. Pooled reference plasma was run prior to and after each batch for quality control and assurance. Plasma sample aliquots (65 μL) were treated with 130 μL acetonitrile (2:1 vol/vol) containing 3.5 μL of an internal isotopic standard mix,10 (link),15 ,17 (link) placed on ice for 30 minutes, and centrifuged for 10 minutes (16,100g at 4°C) to remove protein. The supernatants were loaded onto a Shimadzu autosampler maintained at 4°C and analyzed in triplicate using a Thermo LTQ Velos Orbitrap (Thermo Scientific, San Jose, CA, USA) and C18 column chromatography. Elution was obtained with a formic acid/acetonitrile gradient at a flow rate of 0.35 mL/min for the initial 6 minutes and 0.5 mL/min for the remaining 4 minutes. The first 2-minute period consisted of 5% solution A (2% [vol/vol] formic acid in water), 60% water, 35% acetonitrile, and the final 4-minute period was maintained at 5% solution A, 95% acetonitrile. The mass spectrometer was set to collect mass-to-charge ratio (m/z) from 85 to 2000 over 10 minutes at 60,000 mass resolution. Electrospray ionization was used in positive mode for detection.9 (link),10 (link),15 ,17 (link)