Acyl-CoAs were analyzed on an Ultimate 3000 Quaternary UHPLC coupled to a Q Exactive Plus mass spectrometer operating in the positive ion mode with a heated ESI probe in an IonMax Source housing. Samples were kept in a temperature controlled autosampler at 6 °C and LC separation was performed as previously described on a Waters XBridge 3.5 μm particle size C18 2.1 × 150 mm column. LC conditions were as follows modified from previous studies[22 (link), 20 (link)]; column oven temperature 25 °C, solvent A water with 5 mM ammonium acetate, solvent B 95:5 acetonitrile: water with 5 mM ammonium acetate, solvent C (wash solvent) 80:20 acetonitrile: water with 0.1% formic acid. The gradient was as follows: 0.2 mL/min flow at 98% A and 2% B for 1.5 min, 80% A 20% B at 5 min, 100% B at 12 min, 0.3 mL/min 100% B at 16 min, 0.2 mL/min 100% C at 17 min, held to 21 min, then re-equilibrated at 0.2 mL/min flow at 98% A and 2% B from 22 to 28 min. Flow from 4–18 minutes was diverted to the instrument. Operating conditions on the mass spectrometer were as follows; auxiliary gas 10 arbitrary units (arb), sheath gas 35 arb, sweep gas 2 arb, spray voltage 4.5 kV, capillary temperature 425 °C, S-lens RF-level 50, aux gas heater temperature 400 °C, in-source CID 5 eV. Scan parameters were optimized during the experiments, but final conditions were alternating full scan from 760–1800 m/z at 140,000 resolution and data independent acquisition (DIA) looped 3 times with all fragment ions multiplexed at a normalized collision energy (NCE) of 20 at a resolution of 280,000. An isolation width of 7 m/z with an offset of 3 m/z was used to capture all relevant isotopologues for targeted acyl-CoAs. Data was processed in Xcalibur, TraceFinder (Thermo), and then isotopic enrichment was calculated by the method of Fernandez, et al., to compensate for the non-linearity of isotopic enrichment[23 (link)]. Statistical analysis and graphical plots were generated in Prism v6 (GraphPad, LaJolla, CA). Using these conditions, we re-validated the precision of this LC-MS/HRMS method for acyl-CoAs. We conducted validation for inter- (n=3) and intra-day (n=3) precision for low (LQC), medium (MQC) and high quality control (HQC) levels of 12.5, 50, and 250 ng on column, respectively. Co-efficient of variation (inter-/intra-day) for LQC, MQC, and HQC of acetyl-CoA were 16.9%/16.1%, 2.7%/6.2%, 18.2%/13.5%, respectively. Values for succinyl-, propionyl- and other short chain acyl-CoAs were likewise below 20% for inter- and intra-day variation.