Liquid chromatography–mass spectrometry

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 was successfully used for LC-MS applications between 1978 and 1990 because it allowed coupling of 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. MBI interfaces for LC-MS allowed MS to be 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.[4][7]

The direct liquid introduction (DLI) interface 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 horse 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.[2][4]

The thermospray (TSP) interface 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 comprising the 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).[2][3][7]

The frit FAB and continuous flow-FAB (CF-FAB) interfaces were developed in 1985 and 1986 respectively.[7] 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 a 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.[2][4]

ESI interface for LC-MS systems was developed by Fenn and collaborators in 1988.[15] This ion source/ interface can be used for the analysis of moderately polar molecules (e.g., metabolites, xenobiotics, and peptides). The liquid eluate coming out of the LC column is pumped through a metal capillary kept at 3 to 5 kV. The liquid is nebulized at the tip of the capillary and a fine spray of charged droplets is formed. To avoid contamination, this capillary is usually perpendicularly located at the inlet of the MS system. The heat created by the electric potential is used to rapidly evaporate the droplets in an atmosphere of dry nitrogen. Later, the ionized analytes are transferred into the high vacuum chamber of the MS as the charged ions flow through a series of small apertures with the aid of focusing voltages. Positively and negatively charged ions can be detected and it is possible to switch between the negative and positive modes of operation. Most ions produced in the ESI interface are multiply charged.[3] The use of 1–3 mm ID microbore columns is recommended for LC-MS systems using electrospray ionization (ESI) interfaces because optimal operation is achieved with flow rates in the 50-200 μl/min range.[4]

The development of the APCI interface for LC-MS started with Horning and collaborators in the early 1973.[16] However, its commercial application was introduced at the beginning of the 1990s after Henion and collaborators improved the LC-APCI-MS interface in 1986.[4] The APCI ion source/ interface can be used to analyze small, neutral, relatively non-polar, and thermally stable molecules (e.g., steroids, lipids, and fat soluble vitamins). These compounds are not well ionized using ESI. In addition, APCI can also handle mobile phase streams containing buffering agents. The liquid from the LC system is pumped through a capillary and there is also nebulization at the tip, where a corona discharge takes place. First, the ionizing gas surrounding the interface and the mobile phase solvent are subject to chemical ionization at the ion source. Later, these ions react with the analyte and transfer their charge. The sample ions then pass through small orifice skimmers by means of or ion-focusing lenses. Once inside the high vacuum region, the ions are subject to mass analysis. This interface can be operated in positive and negative charge modes and singly-charged ions are mainly produced.[3] APCI ion source can also handle flow rates between 500 and 2000 μl/min and it can be directly connected to conventional 4.6 mm ID columns.[7]

The APPI interface for LC-MS was developed simultaneously by Bruins and Syage in 2000.[17][4] APPI is another LC-MS ion source/ interface for the analysis of neutral compounds that cannot be ionized using ESI.[3] This interface is similar to the APCI ion source, but instead of a corona discharge, the ionization occurs by using photons coming from a discharge lamp. In the direct-APPI mode, singly charged analyte molecular ions are formed by absorption of a photon and ejection of an electron. In the dopant-APPI mode, an easily ionizable compound (Dopant) is added to the mobile phase or the nebulizing gas to promote a reaction of charge-exchange between the dopant molecular ion and the analyte. The ionized sample is later transferred to the mass analyzer at high vacuum as it passes through small orifice skimmers.[4]

LC-MS is widely used in the field of bioanalysis and is specially involved in pharmacokinetic studies of pharmaceuticals. Pharmacokinetic studies are needed to determine how quickly a drug will be cleared from the body organs and the hepatic blood flow. MS analyzers are useful in these studies because of their shorter analysis time, and higher sensitivity and specificity compared to UV detectors commonly attached to HPLC systems. One major advantage is the use of tandem MS-MS, where the detector may be programmed to select certain ions to fragment. The measured quantity is the sum of molecule fragments chosen by the operator. As long as there are no interferences or ion suppression in LC-MS, the LC separation can be quite quick.[19]

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.[20] 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 (e.g., 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.[19] Recently, LC-MS/MS has been applied to search peptide biomarkers. An example is the recent discovery and validation of peptide biomarkers for four major bacterial respiratory tract pathogens (Staphylococcus aureus, Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae).[21]

LC-MS has emerged as one of the most commonly used techniques in global metabolite profiling of biological tissue (e.g., blood plasma, serum, urine).[22] LC-MS is also used for the analysis of natural products and the profiling of secondary metabolites in plants.[23] 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.[24] 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.[25] 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.[26]

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, lipodomics, natural products dereplication, bioaffinity screening, in vivo drug screening, metabolic stability screening, metabolite identification, impurity identification, quantitative bioanalysis, and quality control.[27]