Liquid Chromatography-Mass Spectrometry (LC-MS)
What is Liquid Chromatography-Mass Spectrometry?
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.
Features of Liquid Chromatography-Mass Spectrometry
- Minimal sample preparation required for rapid analysis and flow-through
- High sensitivity combined with the ability to analyze multiple compounds, even across compound classes.
- High accuracy, high resolution identification and quantification of target analytes.
Applications of Liquid Chromatography-Mass Spectrometry
The coupling of MS with LC systems is attractive because liquid chromatography can separate delicate and complex natural mixtures, which chemical composition needs to be well established (e.g., biological fluids, environmental samples, and drugs). Further, LC-MS has applications in volatile explosive residue analysis. 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. As an example, HPLC-MS is regarded as the leading analytical technique for proteomics and pharmaceutical laboratories. Other important applications of LC-MS include the analysis of food, pesticides, and plant phenols.
1. Pharmacokinetics
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.
2. 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 (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. Recently, LC-MS/MS has been applied to search peptide biomarkers. Examples are the recent discovery and validation of peptide biomarkers for four major bacterial respiratory tract pathogens (Staphylococcus aureus, Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae) and the SARS-CoV-2 virus.
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). 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.
3. 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, lipodomics, natural products dereplication, bioaffinity screening, in vivo drug screening, metabolic stability screening, metabolite identification, impurity identification, quantitative bioanalysis, and quality control.
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