5500 q trap lc ms ms

500 μL of blood was mixed 1:2 with 138 mM NaCl, 8 mM Na₂HPO₄, 2 mM KH₂PO₄, 3 mM KCl, 5.9 mM NaHCO₃, 5.6 mM dextrose, 2.15 mM CaCl₂, 1% DMSO, and 120 μM dipyridamole. Samples were centrifuged at 3650 × g for 10 min at 4° and proteins precipitated by addition of 2% v/v of 70% (12N) perchloric acid with re-centrifuged at 3650 × g for 10 min at 4°C. Adenosine was determined on a 5500 QTRAP LC/MS/MS (AB Sciex, Foster City, CA) with a 1200 series HPLC (Agilent Technologies, Santa Clara, CA). LC separation was performed on a Phenomenex Kinetex 2.6 u PFP column (4.6 × 100mm) (Torrence, CA). Mobile phase A was 0.1% formic acid in water. Mobile phase B was 0.1% formic acid in acetontrile. Flow rate was 0.3 mL/min and the linear gradient was: 0-1 min, 100% A; 5 min, 90% A; 10 min, 80% A; 12-18 min, 0% A; and 18.5-25 min, 100% A with an auto-sampler temperature of 5°C. The injection volume was 5 μL. Mass spectra were acquired with positive electrospray ionization with an ion spray voltage of 5500 V at a source temperature of 450 °C. The curtain gas, ion source gas 1 and ion source gas 2 were 35, 50, and 65, respectively. Multiple reaction monitoring was used to quantify adenosine m/z 268.1 --> m/z 136.1, inosine m/z 269.1 --> m/z 137.1 and internal standard caffeine m/z 195.1 --> m/z 138.1.