Use of Internal Standards in LC-MS Bioanalysis

Internal standards (ISs) are commonly used in liquid chromatography-mass spectrometry (LC-MS) bioanalysis. The main purpose of utilizing ISs is to improve the accuracy and precision of quantitation as well as the robustness of bioanalytical methods. The accuracy and precision of reported concentrations and the reliability of bioanalytical methods can be significantly improved through the proper use of a good IS. Then, what is a good IS? How should its concentration be determined? When and how should it be added? Why are stable isotope labeled ISs preferred yet one should still be cautious in their usage? Should IS responses be monitored during incurred sample analysis? What are the root causes of IS response variations? What are their potential impacts on the integrity of reported concentrations? These questions are addressed in this chapter with a focus on small molecules (molecular weights typically less than 1000 Da).