1260 infinity hplc

HPLC evaluations were performed as previously described¹⁰ . Briefly, extractions were obtained from transwell permeable support inserts containing T84 cells which were submerged in 500 μl of 80% MeOH. The sample was then placed in liquid N₂ until frozen. The extraction process was repeated twice more. The resulting 1.5 ml of extract was dried via an Eppendorf Vacufuge at room temperature. The dried extract was dissolved in 200 μl of HPLC mobile phase A (details below) and filtered (Whatman Puradisc 4, 0.45 μm, nylon) into vials for HPLC injection. Analyses were performed on an Agilent Technologies 1260 Infinity HPLC using a Phenomenex Luna C18(2) column.

Whole murine hearts were collected in 1 mL of 80% MeOH and flash-frozen under liquid nitrogen and stored at −80°C. Adenosine was extracted and quantified in the tissue as described (Lee et al., 2018 (link)). Analyses were performed on an Agilent Technologies 1260 Infinity HPLC using a phenomenex Luna C18(2) column (100 Å, 150 X 4.6 mm) (mobile phase A: 50 mM KH2PO4, 5 mM tetrabuty-lammonium bisulfate, pH 6.25; mobile phase B: acetonitrile; column temperature: 30°C; flow rate: 1 mL/min; 75 μL injection). Samples were filtered through VIVASPIN 500 membranes (Sartorius Stedim Biotech, 5,000 MWCO, PES) prior to HPLC analysis. Chromatographic separation of the metabolites was performed using a combination of isocratic and gradient methods including column washing and equilibration periods at the end (0 min: 100% A; 7 min: 100% A; 10 min: 97% A; 18 min: 97% A; 45 min: 86% A; 60 min: 50% A; 80 min: 50% A; 90 min: 100% A; 135 min: 100% A). Adenosine was detected by absorption at 254 nm, and the absorbance spectra and retention time verified by co-injection with an authentic standard.