Internal standard response variations during incurred sample analysis by LC–MS/MS: Case by case trouble-shooting

Abstract Internal standard (IS) responses can directly impact the accuracy of reported concentrations in bioanalysis as the majority of LC–MS/MS methods are based on analyte/IS response ratios for quantitation. Due to the complexity of incurred sample matrices and drug formulation, variable IS responses are quite common upon applying a validated method to the analysis of incurred samples. To maintain the integrity of a study and to avoid economic losses, it is therefore extremely important to monitor IS response variations during bioanalysis and to quickly identify the root causes if variations are observed. Presented in this article are twelve trouble-shooting examples from the analyses of incurred samples by a wide variety of bioanalytical methods, including human error, malfunctioning equipment/instruments, wrong material, matrix effect and inherent issues with a bioanalytical method. Insightful ideas for how to trouble-shoot and how to develop more reliable bioanalytical methods can be drawn from these practical examples.