How much separation for LC–MS/MS quantitative bioanalysis of drugs and metabolites?

Abstract LC–MS/MS has been the dominant analytical technology for quantitative bioanalysis of drugs and metabolites for more than two decades. Despite this, a very fundamental question like how much separation is required for LC–MS/MS quantitative bioanalysis of drugs and metabolites has not been adequately addressed. Some think that no or only very limited separation is necessary thanks to the unparalleled selectivity offered by tandem mass spectrometry. Others think that the more separation, the better, because of the potential detrimental impact of matrix effect (ion suppression or enhancement). Still others just use a rule-of-thumb approach by keeping the adjusted retention/capacity factor always between 2 and 5. The purpose of this article is to address this fundamental question through rational thinking together with various real case examples drawn from regulated bioanalytical laboratories.