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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 structural identity of the individual components with high molecular specificity and detection sensitivity. 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.

In addition to the liquid chromatography and mass spectrometry devices, an LC-MS system contains an interface that efficiently transfer the separated components from the LC column into the MS ion source. The interface is necessary because 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 vacuum (around 10−6 torr). Thus, it is not possible to directly pump the eluate from the LC column into the MS source. Overall, the interface is 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. Nowadays, most extensively applied LC-MS interfaces are based on atmospheric pressure ionization (API) strategies like electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo-ionization (APPI). These interfaces became available in the 1990s after a two decades long research and development process.

History of LC-MS
The coupling of chromatography with MS is a well developed chemical analysis strategy dating back from the 1950s. Gas chromatography (GC) - MS was originally introduced in 1952, when A.T. James and A.J.P Martin were trying to develop tandem separation - mass analysis techniques. In GC, the analytes are eluted from the separation column as a gas and the connection with electron inization (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. The development of LC-MS systems took longer than GC-MS and was directly related to the development of proper interfaces. V.L. Tal'roze and collaborators started the development of LC-MS in the early 1970s, when they first used capillaries to connect LC columns and MS ion sources. A similar strategy was investigated by McLafferty and collaborators in 1973. 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. However, such analyte off-line treatment process was time consuming and comprised risks 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.

Moving-belt interface
The moving-belt interface (MBI) was developed in 1977. This interface consisted of an endless moving belt receiving the LC column effluent. On the belt, the solvent was evaporated by gently heating and efficiently exhausting the solvent vapors under reduced pressure in two vacuum chambers. After removing the liquid phase, the analytes would desorb from the belt and migrate to the MS ion source to be analysed. MBI interface was successfully used for LC-MS applications between 1978 and 1990 because it allowed to couple LC to MS devices using EI, CI, and fast-atom bombardment (FAB) ion sources. The most common MS systems connected by MBI interfaces to LC columns were magnetic sector and quadropole instruments. By using MBI interfaces for LC-MS, this technique was widely applied in the analysis of drugs, pesticides, steroids, alkaloids, and polycyclic aromatic hydrocarbons. This interface is no longer used because of its mechanical complexity and the difficulties associated to belt renewal. Particle beam interfaces took over the wide applications of MBI for LC-MS in 1988.

Direct liquid introduction interface
The direct liquid introduction interface (DLI) was developed in 1980. This interface was thought as a solution to the evaporation of liquid inside the capillary inlet interface. In DLI, a nebulizer was used to disintegrate part of the effluent coming from the column. A small diaphragm was used to form a liquid jet composed of small droplets that were subsequently dried in a desolvation chamber. A microbore capillary column was used to transfer the nebulized liquid product to the MS ion source. The analytes were ionized using a solvent assisted chemical ionization source, where the LC solvents acted as reagent gases.To use this interface, it was necessary to split the flow coming out of the LC column because only a small portion of the effluent (10 to 50 μl/min out of 1 ml/min) could be analyzed on-line without breaking the MS vacuum. One of the main operational problems of the DLI interface was the frequent clogging of the diaphragm orifices. The DLI interface was used between 1982 and 1985 for the analysis of pesticides, corticosteroids, metabolites in equine urine, erythromycin, and vitamin B12. However, this interface was replaced by the thermospray interface, which removed the flow rate limitations and the issues with the clogging diaphragms.

Thermospray interface
Te thermospray interface (TSP) was developed in 1983 by Vestal laboratories at the University of Houston. 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 by a heated probe, a desolvation chamber, and an ion exchange 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. The ionization of solutes occurred by direct evaporation or ion-molecule reactions induced by the solvent. This interface was able to handle up to 2 ml/min of eluate from the LC column and would efficiently introduce it into the MS vacuum system. TSP was also more suitable for LC-MS applications involving reversed phase liquid chromatography (RT-LC). The TSP system had a dual function acting as an interface and a solvent-mediated chemical ionization source. With time, the mechanical complexity of TSP was simplified, and this interface became popular as the first ideal LC-MS interface for pharmaceutical applications, i.e., analysis of drugs, metabolites, conjugates, nucleosides, peptides, natural products, and pesticides. The introduction of TSP marked a significant improvement for LC-MS systems and was the most widely applied interface until the beginning of the 1990s, when it began to be replaced by interfaces involving atmospheric pressure ionization (API).

FAB Based Interfaces
The frit FAB and continuous flow-FAB (CF-FAB) interfaces were developed in 1985 and 1986 respectively. Both interfaces were similar, but they differed in that the first used a porous frit probe as connecting channel, while CF-FAB used a probe tip. From these, the CF-FAB was more successful as a LC-MS interface and was useful to analyze non-volatile and thermally labile compounds. In these interfaces, the LC effluent passed through the frit or CF-FAB channels to form an uniform liquid film at the tip. There, the liquid was bombarded with ion beams or high energy atoms (fast atom). For stable operation, the FAB based interfaces were able to handle liquid flow rates of only 1-15 μl and were also restricted to microbore and capillary columns. In order to be used in FAB MS ionization sources, the analytes of interest should be mixed with a matrix (e.g., glycerol) that could be added before or after the separation in the LC column. FAB based interfaces were extensively used to characterize peptides, but lost applicability with the advent of electrospray based interfaces in 1988.





Liquid chromatography
Present day liquid chromatography generally utilizes very small particles packed and operating at relatively high pressure, and is referred to as high performance liquid chromatography (HPLC); modern LC-MS methods use HPLC instrumentation, essentially exclusively, for sample introduction. In HPLC, the sample is forced by a liquid at high pressure (the mobile phase) through a column that is packed with a stationary phase generally composed of irregularly or spherically shaped particles chosen or derivatized to accomplish particular types of separations. HPLC methods are historically divided into two different sub-classes based on stationary phases and the corresponding required polarity of the mobile phase. Use of octadecylsilyl (C18) and related organic-modified particles as stationary phase with pure or pH-adjusted water-organic mixtures such as water-acetonitrile and water-methanol are used in techniques termed reversed phase liquid chromatography (RP-LC). Use of materials such as silica gel as stationary phase with neat or mixed organic mixtures are used in techniques termed normal phase liquid chromatography (NP-LC). RP-LC is most often used as the means to introduce samples into the MS, in LC-MS instrumentation.

Mass spectrometry
Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. MS works by ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios.[1] In a typical MS procedure, a sample is loaded onto the MS instrument and undergoes vaporization. The components of the sample are ionized by one of a variety of methods (e.g., by impacting them with an electron beam, using uv lights as a photon beam, laser beam or corona discharge etc.), which results in the formation of charged particles (ions). The ions are separated according to their mass-to-charge ratio in an analyzer by electromagnetic fields. The ions are detected, usually by a quantitative method. The ion signal is processed into mass spectra.

Additionally, MS instruments consist of three modules. An ion source, which can convert gas phase sample molecules into ions (or, in the case of electrospray ionization, move ions that exist in solution into the gas phase). A mass analyzer, which sorts the ions by their masses by applying electromagnetic fields. A detector, which measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present

The technique has both qualitative and quantitative uses. These include identifying unknown compounds, determining the isotopic composition of elements in a molecule, and determining the structure of a compound by observing its fragmentation. Other uses include quantifying the amount of a compound in a sample or studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in a vacuum). MS is now in very common use in analytical laboratories that study physical, chemical, or biological properties of a great variety of compounds.

Mass analyzer
There are many different mass analyzers that can be used in LC/MS. Single quadrupole, triple quadrupole, ion trap, time of flight (TOF) and quadrupole-time of flight (Q-TOF).

Interface
Understandably the interface between a liquid phase technique which continuously flows liquid, and a gas phase technique carried out in a vacuum was difficult for a long time. The advent of electrospray ionization changed this. The interface is most often an electrospray ion source or variant such as a nanospray source; however atmospheric pressure chemical ionization interface is also used. Various deposition and drying techniques have also been used such as using moving belts; however the most common of these is off-line MALDI deposition. A new approach still under development called Direct-EI LC-MS interface, couples a nano HPLC system and an electron ionization equipped mass spectrometer.

Pharmacokinetics
LC-MS is very commonly used in pharmacokinetic studies of pharmaceuticals and is thus the most frequently used technique in the field of bioanalysis. These studies give information about how quickly a drug will be cleared from the hepatic blood flow, and organs of the body. MS is used for this due to high sensitivity and exceptional specificity compared to UV (as long as the analyte can be suitably ionised), and short analysis time.

The major advantage MS has is the use of tandem MS-MS. The detector may be programmed to select certain ions to fragment. The process is essentially a selection technique, but is in fact more complex. The measured quantity is the sum of molecule fragments chosen by the operator. As long as there are no interferences or ion suppression, the LC separation can be quite quick.

Proteomics/metabolomics
LC-MS is also used in proteomics where again components of a complex mixture must be detected and identified in some manner. The bottom-up proteomics LC-MS approach to proteomics generally involves protease digestion and denaturation (usually trypsin as a protease, urea to denature tertiary structure and iodoacetamide to cap cysteine residues) followed by LC-MS with peptide mass fingerprinting or LC-MS/MS (tandem MS) to derive sequence 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 spectrometer. Samples of complex biological fluids like human serum may be run in a modern LC-MS/MS system and result in over 1000 proteins being identified, provided that the sample was first separated on an SDS-PAGE gel or HPLC-SCX.

Profiling of secondary metabolites in plants or food like phenolics can be achieved with liquid chromatography–mass spectrometry.

Drug development
LC-MS is frequently used in drug development at many different stages including peptide mapping, glycoprotein mapping, natural products dereplication, bioaffinity screening, in vivo drug screening, metabolic stability screening, metabolite identification, impurity identification, quantitative bioanalysis, and quality control.