Luna 3 mm nh2 100 a

Neonatal hearts were excised and weighed. Hearts were submerged in 500 μl of extraction solution (80% methanol/10 µM trifluoromethanosulfanate) contained in 2 mL tubes containing 1.4 mm ceramic beads. Tubes were placed in a Bead Mill 4 (Fisher brand) to homogenize tissue for 6 min (speed: 6 m/s). Homogenates were transferred to clean tubes and centrifuged at 4 °C for 10 min at 17,000g. For every sample, a supernatant volume that is equivalent to 5 mg of total heart tissue was transferred to a glass vial. Samples in glass vials were dried under an EZ-2Elite evaporator. Metabolites were resuspended in 100 μl 50% acetonitrile (ACN). The analysis was performed on a Q Exactive (Thermo Scientific) in polarity-switching mode with positive voltage 3.5 kV and negative voltage 3.5 kV. The mass spectrometer was coupled to an Vanquish (Thermo Scientific) UHPLC system. Mobile phase A was 5 mM NH₄Ac, pH 9.9, B was ACN. Separation was achieved on a Luna 3 mm NH₂ 100 A (150 × 2.0 mm) (Phenomenex) column kept at a temperature of 40 °C. Injection volume was 10 μl, flow was 200 μl/min, and the gradient ran from 15% A to 95% A in 18 min, followed by an isocratic step for 9 min and re-equilibration for 7 min. Metabolites were detected and quantified as the area under the curve based on retention time and accurate mass (≤ 15 p.p.m.) using MZMine 2.0.