Sample Preparation for LC‐MS Bioanalysis of Peptides

Quantitative bioanalysis of peptides is becoming more and more important with the continuously increasing interest of peptides in biomedical and pharmaceutical research. LC-MS, due to its high sensitivity and selectivity, has become the preferred method for the quantitative bioanalysis of peptides, including both peptide therapeutics and endogenous peptides. However, development of a LC-MS bioanalytical method for peptides remains challenging. Sample preparation is a critical, and often the most challenging, part in the development of a LC-MS bioanalytical method. It plays an important role in ensuring the selectivity, sensitivity, reproducibility and robustness of the method. Various sample preparation strategies have been developed for the extraction of peptides. The commonly used techniques include protein precipitation (PPT), liquid-liquid extraction (LLE), solid-phase extraction (SPE), and immunoaffinity extraction (IAE). These strategies will be introduced and discussed in details in this chapter. Other less commonly used techniques, including immobilized metal ion affinity chromatography (IMAC), molecular imprinted polymers (MIPs), ultrafiltration, derivatization and digestions, will also be introduced. In addition, sample pretreatment is often required prior to sample extraction to prevent the adsorption and ensure the integrity of the peptide analytes during sample collection, processing and storage. In this chapter, various sample pretreatment considerations and strategies will also be introduced and described.