Liquid Chromatography/Mass Spectrometry

Abstract

AbstractLiquid chromatography/mass spectrometry (LC/MS) is the combination of high‐performance liquid chromatography (HPLC) and mass spectrometry (MS) into a single, continuous‐flow analytical system. HPLC separates analytes from their matrix and from one another in a flowing system on a time basis. These separated analytes are removed from the solvent stream and introduced to the MS system through one of several types of available interfaces which facilitate ionization of the analyte molecules. MS is an analytical technique which separates these analyte ions on the basis of their mass‐to‐charge ratio (m/z). Data collected from LC/MS is a series of mass spectra taken over time as the chromatographic effluent exits the column and is analyzed by the mass spectrometer. The three interface types in common usage are particle beam (PB), atmospheric pressure chemical ionization (APCI) and electrospray interface (ESI). These interfaces differ in the characteristics of the mass spectra that they produce, the types of chemical compounds for which they are best suited and the interferences to which they are subject. With the combined capability of two of these interfaces, APCI and ESI, LC/MS is able to address nearly any type of organic molecule from small molecules to biological macromolecules. ESI has ushered in a revolution in biochemical research on large molecules although the technology itself is not completely understood and is subject to a variety of interferences. APCI is limited to smaller molecules and is most at home in routine use for very high‐throughput drug and metabolite analyses. The PB interface, although not as flexible as the other two, is able to produce electron ionization (EI) spectra which can be searched against standard, commercial libraries. ESI and APCI are not able to produce EI spectra but, through the use of collisionally activated dissociation (CAD), are able to produce usable spectra in both single‐stage and tandem mass spectrometry (MS/MS).