Abstract
Biomarkers, or more specifically molecular markers, can detect biochemical changes associated with disease processes and drug effects before histopathological and pathophysiological changes occur. Multiplexing technologies such as high-performance liquid chromatography/mass spectrometry (LC-MS) allow for the measurement of molecular marker patterns that confer significantly more information than the measurement of a single parameter alone. The use of multiplexing assays for drug development, and as diagnostic tools, is attractive but will require regulatory review and approval and thus requires validation following regulatory guidances. Multiplexing assays always constitute a compromise. The number of analytes that can reasonably be included in a mass spectrometry-based multiplexing assay depend on the physico-chemical properties of the analytes and their integration into a single assay in terms of extraction, HPLC separation, ionization conditions and mass spectrometry detection. Another aspect includes biomedical considerations such as the differences in physiological concentrations of analytes, the required concentration range, and how much variability is acceptable before the clinical utility of a marker is negatively affected. Regulatory considerations include validation and quality control during sample analysis. Current bioanalytical regulatory guidelines have mostly been developed for single drug compounds and are not always adequate for multiplexing molecular marker assays that often quantify endogenous compounds. Specific guidances for multiplexing assays should be developed. Even if it is possible to integrate a wide variety and large number of analytes into a multiplexing assay, it should always be taken into consideration that a set of shorter, more specialized assays, may offer a more manageable and efficient alternative.