This chapter gives an introduction into the role of mass spectrometry in the structure elucidation of small molecules. It provides an overview of important aspect in acquiring data using mass spectrometry, especially in on‐line combination with gas or liquid chromatography. For clarity, the discussion is limited to the use of either electron ionisation or electrospray ionisation. Concepts of mass analysis and tandem mass spectrometry are discussed, as well possible data acquisition strategies to assist in structure elucidation. Some typical fragmentation and mass spectral interpretation issues are highlighted.
To illustrate the potential of (tandem) mass spectrometry in structure elucidation, various examples from different application areas are given. In this respect, attention is paid to confirmation of identity using library searching, multiple transitions in selected‐reaction monitoring, and interpretation of accurate mass and isotope pattern data. The structure elucidation of unknowns is illustrated with examples involved identification of impurities and metabolites of drugs (related substances) and the identification of real unknowns. The examples are given to highlight important steps in the analytical strategies used in structure elucidation by mass spectrometry.