Theory-guided efficient strategy to maximize speed and resolution in rapid gradient LC–MS/MS bioanalysis

Reversed-phase gradient LC–MS/MS bioanalytical methods of 5–100% organic solvent in a 1–3 min gradient time are common in today's bioanalytical laboratory. The goal of this work was to develop a theory-guided systematic strategy for maximizing resolution and speed in rapid gradient LC–MS/MS bioanalysis. We studied the effect of gradient time (tG), initial and final eluent strength (% B = % organic), and flow rate (F) on the separation of multiple critical pairs (Rs) and peak capacity (nc) in a gradient elution of a mixture of five structurally related compounds. By optimizing the gradient time tG, the initial and final percentages of the organic component of the mobile phase, comparable resolution and peak capacity could be achieved in a shorter run time. More importantly, we demonstrated that higher flow rates improved resolution, peak capacity and reduced run time in rapid gradient separations on a 5 μm particle column. A straightforward mathematical explanation of the phenomenon was provided applying basic resolution equations in gradient elution theory. A systematic approach to execute a rapid gradient LC–MS/MS bioanalytical method to shorten run time and improve resolution is proposed, taking into consideration not only the analytes of interest but also potential matrix effects from the dosing vehicle and biological matrix.