Liquid chromatography separates compounds before mass analysis according to how they partition between the mobile and stationary phases. This reduces the number of molecules entering the mass spectrometer at the same time, helping distinguish components in complex biological samples. The resulting separation supports more reliable identification and quantification of metabolites, lipids, peptides, proteins, and other biomolecules.
A precursor ion is selected according to its mass-to-charge ratio and then fragmented inside the instrument. The resulting product ions provide a characteristic pattern that supports molecular identification. Measuring these fragments also contributes to selective quantification, because the analysis evaluates both the original ion and its fragmentation products rather than relying on mass alone.
Targeted analysis focuses on selected molecules and measures them specifically, making it useful when researchers want to assess defined metabolites, lipids, peptides, or other biomolecules. Untargeted analysis surveys molecular features more broadly to help characterize biological changes and discover candidates for further study. The choice depends on whether the investigation begins with defined targets or exploratory biological questions.
LC-MS/MS analysis can be applied to diverse biomolecular classes, including metabolites, lipids, peptides, proteins, and other molecules present in biological samples. This breadth allows one analytical platform to support investigations at several biological levels, from molecular products and lipid composition to peptide or protein changes associated with physiology, disease, or treatment.
The workflow begins with liquid chromatographic separation of compounds in a biological sample. The separated molecules are then ionized, and the mass spectrometer selects precursor ions by mass-to-charge ratio. Those ions are fragmented, and the instrument measures the resulting product ions. Researchers use the combined separation and spectral information to identify and quantify molecular components.
Researchers use this approach to characterize molecular pathways, compare physiological and disease states, identify biomarkers, and measure treatment-related molecular changes. Its high sensitivity and selectivity are valuable when biological samples contain many chemically different components. Results can reveal which molecules change between conditions and can connect those changes with pathways, disease processes, or responses to treatment.