Advances in the quantitation of therapeutic insulin analogues by LC-MS/MS.

Insulin analogues represent a major and growing class of biotherapeutics, and their quantitation is an important focus of commercial and public effort across a number of different fields. As LC-MS has developed, it has become an increasingly practicable and desirable alternative to ligand-binding-based approaches for quantitation of this class of compounds. The sensitivity challenge of measuring trace levels of this large peptide molecule in a protein-containing matrix is considerable; however, different approaches to detection, extraction and separation are described to overcome this challenge, including immunoaffinity capture, SPE and low-flow HPLC. Considerations such as bioanalytical assay acceptance criteria and antidrug antibody effects during drug development are included, alongside descriptions of recent sports doping and clinical applications. Factors affecting the correlation and agreement of MS with biological ligand-binding methods are discussed, with ways to anticipate and appreciate differences between the values derived from each technique. The 'future perspective' section discusses the likely trend towards MS-based analysis for these compounds and the impact of HRMS. A high degree of scientific creativity, combined with science-defined regulatory approaches that define suitable validation criteria, will be needed to meet the demanding requirements for high-throughput analysis of insulin by LC-MS.