Development and application of a LC-MS/MS method to quantify basal adenosine concentration in human plasma from patients undergoing on-pump CABG surgery}

A sensitive and robust LC-MS/MS method was developed to quantify basal adenosine concentrations in human plasma of patients undergoing on-pump coronary artery bypass grafting (CABG) surgery. A strong cation exchange (SCX) monolithic cartridge was used to enrich analyte, improve robustness, and reduce biological complexity. A simple modifier-free mobile phase was employed to improve sensitivity and reproducibility. This method exhibits consistent precision and accuracy, and the RSDs or REs of all the intraday and interday determinations were within 10%. The calibration curve was linear across the examined dynamic range from 1nM to 500nM (r(2)=0.996). LOD and LOQ were determined to be 0.257nM and 0.857nM respectively, while LLOQ was below 10nM. This method was used to monitor changes of adenosine levels in patient plasma drawn intraoperatively during on-pump CABG surgery. The analysis of 84 patients revealed that the mean concentration of adenosine in coronary sinus plasma after cardiopulmonary bypass (CPB) is higher than that in coronary sinus before CPB (p=0.0024; two-tailed t-test) and that in radial artery plasma after CPB (p=0.0409; two-tailed t-test). These findings suggest that the equilibrium between adenosine production and elimination has favored the elevation of adenosine basal level during on-pump CABG surgery and the change is specific to heart tissues. Evaluation of adenosine with a sensitive and robust analytical method has important implications on providing consistent results and meaningful insights into adenosine regulation, as well as its steady state and sustained action on the heart. Relating patient characteristics or clinical outcomes with basal adenosine concentration can be used to optimize the CABG-CPB maneuver by regulating adenosine level via pharmacological intervention, and differentiating adenosine's contribution to cardioprotection from other modulatory factors.