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 that 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 are a series of mass spectra taken over time as the chromatographic effluent exits the column and is analyzed by the mass spectrometer. Although there are several different LC/MS interfaces, the two most commonly used are 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 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 and nonvolatile thermally labile small molecules, although the technology itself is not completely understood and is subject to a variety of interferences. APCI is limited to smaller molecules that are not thermally labile and is most at home in routine use for very high throughput drug and metabolite analyses. A third interface, atmospheric pressure photoionization (APPI), has been recently introduced. APPI operates in a similar manner to APCI and shares the limitation of addressing only small molecules with the additional capability to ionize nonpolar compounds. Finally, combined sources such as APCI/APPI and APCI/ESI have been introduced to improve ease of use and information content in LC/MS analyses.ESI, APCI, and APPI are considered to be soft ionization techniques. This means that the ions formed, which represent the intact molecule, do not produce EI (electron ionization)‐type spectra exhibiting extensive fragmentation of the analyte. These spectra differ therefore from gas chromatography/mass spectrometry (GC/MS)‐produced EI spectra, which can be used to search against standardized compound libraries to facilitate the identification of unknowns. However, through the use of collisionally activated dissociation (CAD), LC/MS is able to produce spectra exhibiting fragmentation patterns in both single‐stage MS and multistage mass spectrometry (MSn). These fragmentation patterns can be used to understand the structure of the analyte and are being used to develop standardized libraries using new computer algorithms. It is important to note that the termcollision‐induced dissociation(CID) is a synonym for CAD. In today's literature, CID is in wider use than CAD; however, the article uses CAD because it more accurately describes the process. As a result of an inelastic collision between an analyte ion and an inert molecule (N2) or atom (Ar, Ne, Xe, or He), the ion's kinetic energy will be translated into internal energy (collisional activation). The ion desiring to reach a lower energy state will subsequently dissociate, thus the termCAD. The ion is not broken apart by the collision, as is implied by the CID term.