Liquid chromatography–mass spectrometry (LC–MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry (MS). Coupled chromatography–MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides spectral information that may help to identify (or confirm the suspected identity of) each separated component. MS is not only sensitive, but provides selective detection, relieving the need for complete chromatographic separation. LC–MS is also appropriate for metabolomics because of its good coverage of a wide range of chemicals. This tandem technique can be used to analyze biochemical, organic, and inorganic compounds commonly found in complex samples of environmental and biological origin. Therefore, LC–MS may be applied in a wide range of sectors including biotechnology, environment monitoring, food processing, and pharmaceutical, agrochemical, and cosmetic industries. Since the early 2000s, LC–MS (or more specifically LC–MS/MS) has also begun to be used in clinical applications.
In addition to the liquid chromatography and mass spectrometry devices, an LC–MS system contains an interface that efficiently transfers the separated components from the LC column into the MS ion source. The interface is necessary because the LC and MS devices are fundamentally incompatible. While the mobile phase in a LC system is a pressurized liquid, the MS analyzers commonly operate under high vacuum. Thus, it is not possible to directly pump the eluate from the LC column into the MS source. Overall, the interface is a mechanically simple part of the LC–MS system that transfers the maximum amount of analyte, removes a significant portion of the mobile phase used in LC and preserves the chemical identity of the chromatography products (chemically inert). As a requirement, the interface should not interfere with the ionizing efficiency and vacuum conditions of the MS system. In GC, the analytes are eluted from the separation column as a gas and the connection with electron ionization (EI) or chemical ionization (CI) ion sources in the MS system was a technically simpler challenge. Because of this, the development of GC-MS systems was faster than LC–MS and such systems were first commercialized in the 1970s. when they first used capillaries to connect an LC column to an EI source. which allowed a higher liquid flow into the source. This was the first and most obvious way of coupling LC with MS, and was known as the capillary inlet interface. This pioneer interface for LC–MS had the same analysis capabilities of GC-MS and was limited to rather volatile analytes and non-polar compounds with low molecular mass (below 400 Da). In the capillary inlet interface, the evaporation of the mobile phase inside the capillary was one of the main issues. Within the first years of development of LC–MS, on-line and off-line alternatives were proposed as coupling alternatives. In general, off-line coupling involved fraction collection, evaporation of solvent, and transfer of analytes to the MS using probes. Off-line analyte treatment process was time-consuming and there was an inherent risk of sample contamination. Rapidly, it was realized that the analysis of complex mixtures would require the development of a fully automated on-line coupling solution in LC–MS.
The key to the success and widespread adoption of LC–MS as a routine analytical tool lies in the interface and ion source between the liquid-based LC and the vacuum-base MS. The following interfaces were stepping-stones on the way to the modern atmospheric-pressure ionization interfaces, and are described for historical interest.
Moving-belt interface
The moving-belt interface (MBI) was developed by McFadden et al. in 1977 and commercialized by Finnigan. This interface consisted of an endless moving belt onto which the LC column effluent was deposited in a band. On the belt, the solvent was evaporated by gently heating and efficiently exhausting the solvent vapours under reduced pressure in two vacuum chambers. After the liquid phase was removed, the belt passed over a heater which flash desorbed the analytes into the MS ion source. One of the significant advantages of the MBI was its compatibility with a wide range of chromatographic conditions. Particle beam interfaces took over the wide applications of MBI for LC–MS in 1988. The PBI operated by using a helium gas nebulizer to spray the eluant into the vacuum, drying the droplets and pumping away the solvent vapour (using a jet separator) while the stream of monodisperse dried particles containing the analyte entered the source. It was commercialized by Vestec and several of the major mass spectrometer manufacturers. The interface resulted from a long-term research project intended to find a LC–MS interface capable of handling high flow rates (1 ml/min) and avoiding the flow split in DLI interfaces. The TSP interface was composed of a heated probe, a desolvation chamber, and an ion focusing skimmer. The LC effluent passed through the heated probe and emerged as a jet of vapor and small droplets flowing into the desolvation chamber at low pressure. Initially operated with a filament or discharge as the source of ions (thereby acting as a CI source for vapourized analyte), it was soon discovered that ions were also observed when the filament or discharge was off. This could be attributed to either direct emission of ions from the liquid droplets as they evaporated in a process related to electrospray ionization or ion evaporation, or to chemical ionization of vapourized analyte molecules from buffer ions (such as ammonium acetate). The fact that multiply-charged ions were observed from some larger analytes suggests that direct analyte ion emission was occurring under at least some conditions. The basic components of a mass spectrometer are the ion source, the mass analyzer, the detector, and the data and vacuum systems. The ion source is where the components of a sample introduced in a MS system are ionized by means of electron beams, photon beams (UV lights), laser beams or corona discharge. In the case of electrospray ionization, the ion source moves ions that exist in liquid solution into the gas phase. The ion source converts and fragments the neutral sample molecules into gas-phase ions that are sent to the mass analyzer. While the mass analyzer applies the electric and magnetic fields to sort the ions by their masses, the detector measures and amplifies the ion current to calculate the abundances of each mass-resolved ion. In order to generate a mass spectrum that a human eye can easily recognize, the data system records, processes, stores, and displays data in a computer. MS is now in very common use in analytical laboratories that study physical, chemical, or biological properties of a great variety of compounds. Among the many different kinds of mass analyzers, the ones that find application in LC–MS systems are the quadrupole, time-of-flight (TOF), ion traps, and hybrid quadrupole-TOF (QTOF) analyzers. A new approach still under development, called direct-EI LC–MS interface, couples a nano HPLC system and an electron ionization equipped mass spectrometer.
Electrospray ionization (ESI)
ESI interface for LC–MS systems was developed by Fenn and collaborators in 1988. This ion source/ interface can be used for the analysis of moderately polar and even very polar molecules (such as metabolites, xenobiotics, peptides, nucleotides, and polysaccharides). The liquid eluate coming out of the LC column is directed into a metal capillary kept at 3 to 5 kV and is nebulized by a high-velocity coaxial flow of gas at the tip of the capillary, creating a fine spray of charged droplets in front of the entrance to the vacuum chamber. To avoid contamination of the vacuum system by buffers and salts, this capillary is usually perpendicularly located at the inlet of the MS system, in some cases with a counter-current of dry nitrogen in front of the entrance through which ions are directed by the electric field. In some sources, rapid droplet evaporation and thus maximum ion emission is achieved by mixing an additional stream of hot gas with the spray plume in front of the vacuum entrance. In other sources, the droplets are drawn through a heated capillary tube as they enter the vacuum, promoting droplet evaporation and ion emission. These methods of increasing droplet evaporation now allow the use of liquid flow rates of 1–2 mL/min to be used while still achieving efficient ionisation and high sensitivity. As a result, while the use of 1–3 mm microbore columns and lower flow rates of 50–200 μl/min was commonly considered necessary for optimum operation in the past, this limitation is no longer as important, and the higher column capacity of larger bore columns can now be advantageously employed with ESI LC–MS systems. Positively and negatively charged ions can be created by switching polarities, and it is possible to acquire alternate positive and negative mode spectra rapidly within the same LC run. While most large molecules (greater than MW 1500–2000) produce multiply charged ions in the ESI source, the majority of smaller molecules produce singly charged ions. However, its commercial application was introduced at the beginning of the 1990s after Henion and collaborators improved the LC–APCI–MS interface in 1986. Nowadays, LC–MS has become one of the most widely used chemical analysis techniques because more than 85% of natural chemical compounds are polar and thermally labile, and GC-MS cannot process these samples.
Proteomics/metabolomics
LC–MS is used in proteomics as a method to detect and identify the components of a complex mixture. The bottom-up proteomics LC–MS approach generally involves protease digestion and denaturation using trypsin as a protease, urea to denature the tertiary structure, and iodoacetamide to modify the cysteine residues. After digestion, LC–MS is used for peptide mass fingerprinting, or LC–MS/MS (tandem MS) is used to derive the sequences of individual peptides. LC–MS/MS is most commonly used for proteomic analysis of complex samples where peptide masses may overlap even with a high-resolution mass spectrometry. Samples of complex biological material, such as human serum, may be analyzed in modern LC–MS/MS systems, which can identify over 1000 proteins. However, this high level of protein identification is possible only after separating the sample by means of SDS-PAGE gel or HPLC-SCX.
LC–MS has emerged as one of the most commonly used techniques in global metabolite profiling of biological tissue such as blood plasma, serum, and urine. LC–MS is also used for the analysis of natural products and the profiling of secondary metabolites in plants. In this regard, MS-based systems are useful to acquire more detailed information about the wide spectrum of compounds from a complex biological samples. LC–nuclear magnetic resonance (NMR) is also used in plant metabolomics, but this technique can only detect and quantify the most abundant metabolites. LC–MS has been useful to advance the field of plant metabolomics, which aims to study the plant system at molecular level providing a non-biased characterization of the plant metabolome in response to its environment. The first application of LC–MS in plant metabolomics was the detection of a wide range of highly polar metabolites, oligosaccharides, amino acids, amino sugars, and sugar nucleotides from Cucurbita maxima phloem tissues. Another example of LC–MS in plant metabolomics is the efficient separation and identification of glucose, sucrose, raffinose, stachyose, and verbascose from leaf extracts of Arabidopsis thaliana.
Drug development
LC–MS is frequently used in drug development because it allows quick molecular weight confirmation and structure identification. These features speed up the process of generating, testing, and validating a discovery starting from a vast array of products with potential application. LC–MS applications for drug development are highly automated methods used for peptide mapping, glycoprotein mapping, lipidomics, natural products dereplication, bioaffinity screening, in vivo drug screening, metabolic stability screening, metabolite identification, impurity identification, quantitative bioanalysis, and quality control.
See also
- Gas chromatography–mass spectrometry
- Capillary electrophoresis–mass spectrometry
- Ion-mobility spectrometry–mass spectrometry