Sample Preparation Methods for Targeted Biomarker Quantification by LC‐MS

Sample preparation is an essential component for bioanalytical methodologies. This chapter elaborates and discusses the current methodologies of sample preparation for both small molecules and macromolecules, with an emphasis on issues of sensitivity, selectivity, and robustness. It also discusses several common issues first, followed by the rationale, features, and limitations of individual methods. The chapter then illustrates applications of the methods. For small molecule biomarkers, protein precipitation (PPT), liquid-liquid extraction (LLE), and solid-phase extraction (SPE) techniques has been regarded as effective means for the majority of biomarkers analyzed. For targeted quantification of protein biomarkers, it is important to establish optimal protocols for sample cleanup and proteolytic digestion of the target protein that achieve high and reproducible recovery of the signature peptides (SPs). LC-MS-based protein quantification, mostly employing selected reaction monitoring (SRM), is based on the detection of signals from SPs released from the native proteins.