Using temperature to optimize resolution and reduce analysis times for bioanalytical diastereomer LC-MS/MS separations.

A rugged and reproducible liquid chromatographic tandem mass spectrometric bioanalytical method was developed for the quantitation of drug stereoisomers in human plasma. Column temperature was shown to be an important variable toward optimizing diastereomer selectivity, resolution and analysis cycle time. Non-linear Van't Hoff plots and changes in peak shape with temperature suggested that selectivity was governed by multiple retention mechanisms. The high temperature chromatography method was validated and used to analyze samples from human clinical trials. Utilization of high temperature chromatography offered alternative selectivity and is a viable approach for difficult separations in regulated bioanalysis.