ADVANCED CHROMATOGRAPHY AND MASS SPECTROMETRY TECHNIQUES FOR THE ANALYSIS OF BIOACTIVE CONSTITUENTS IN FOOD AND CLINICAL FIELDS

The complete set of lipids in an organism or a cell along with its interactions with other molecules, such as other lipids, proteins, and metabolites constitutes the lipidome. Lipidomics is the comprehensive and quantitative study of the lipidome. It involves identification and quantitation of thousands of biological pathways involving lipids and their interactions. Lipids are the essential metabolites in human body; their main biological functions are the energy storage, endocrine actions, morphogenesis, building blocks of cellular and subcellular membranes and signaling molecules. The dysregulation of lipids is related to various serious human diseases, such as cancer, Alzheimer, cardiovascular diseases, and lysosomal disorders. Using lipidomics approaches, it has become easier to study the lipids species in an organism. Lipidomics is an emerging field in the name of the ‘omics’ for system-level analysis of lipids and their interacting partners within a cell. Lipidomics aims to define and quantitate all of the molecular lipid species present in a cell. The lipid molecular species can be described by the eight known categories of lipids, numerous classes, and subclasses, such us fatty acids, glycerolipids, sphingolipids, prenols, sterols, glycerophospholipids, poliketides and saccharolipides. Current studies related to lipid identification and determination, or lipidomics in biological samples, are one of the most important issues in modern bioanalytical chemistry. There are many articles dedicated to specific analytical strategies and to the actual analytical methodologies used in lipidomics in various kinds of biological samples. The most important methods used to characterize the lipidomics in modern bioanalysis are: chromatography/separation methods (thin layer chromatography (TLC), (ultra) high-pressure liquid chromatography ((U)HPLC), gas chromatography (GC), (ultra-performance) supercritical fluid chromatography ((UHP)SFC), and capillary electrophoresis (CE)); spectroscopic methods (Raman spectroscopy (RS), Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR)); mass spectrometry and also hyphenated methods (matrix-assisted laser desorption/ionization (MALDI), hyphenated methods, which include liquid chromatography–mass spectrometry (LC-MS), gas chromatography–mass spectrometry (GC-MS) and also multidimensional techniques). These are being used to identify and quantify all the lipid species in order to understand their function in biological systems. MS technology has been proved to be highly efficient in the characterization and quantification of lipid molecular species in lipid extracts. One of the reasons behind this could be the ability of MS to characterize and separate each ionized particle according to their mass-to-charge (m/z) ratio. MS can also provide structural information by fragmenting the lipid ions which can be achieved by using tandem MS, or MS/MS. Basically, there are two different approaches for lipidomics analysis: - to apply some extraction protocols optimized for each lipid category, and then subject to LC to separate the present lipids molecular species optimally, then the LC eluate is coupled directly to the mass spectrometer for further analysis such as molecular fragmentation (MS/MS), ion scanning, etc. - another approach, also known as “shotgun lipidomics”, involves the offline extraction of lipids followed by MS analysis without LC separation. Several tools are available for lipidomics and some are emerging concerning the combination of genomics and lipidomics to identify clinically relevant biomarkers. For example, SimLipid is a high-throughput characterization tool for lipids. It analyzes lipid mass spectrometric data to profile them using LC coupled with MALDI-MS, MS/MS data, and also remove the overlapping isotopic peaks from multiple spectra in batch mode. "Lipidomics" applies to studying lipid metabolism on a broad scale and it may elucidate the biochemical mechanism(s) underlying specific changes in lipid metabolism. Advances in mass spectrometry have greatly accelerated the lipidomics field. Chemical derivatization has shown its broad use in improving analytical sensitivity and specificity in lipidomics. Lipidomics aims to quantitatively define lipid classes, including their molecular species, in biological systems and it has experienced rapid progress, mainly because of continuous technical advances in instrumentation that are now enabling quantitative lipid analyses with an unprecedented level of sensitivity and precision. The still-growing category of lipids includes a broad diversity of chemical structures with a wide range of physicochemical properties. Reflecting this diversity, different methods and strategies are being applied to the quantification of lipids. Since its advent, LC is being exploited by separation scientists and applied to a wider and wider range of sample matrices for the separation, identification and quantification of ever more compounds, particularly in lipidomic analysis. The unceasing progresses in column and stationary phases production, and the enormous developments in detection techniques have contributed to the outstanding success of chromatography, as an invaluable tool in analytical chemistry in many different fields including nutraceutical, food, environmental, clinical, forensic, and pharmaceutical applications. The great advantages to be gained by the use of LC are especially increased with the hyphenation to MS (LC-MS). Recent trends in the area of LC-MS and related techniques involve: (a) the shift from conventional HPLC-MS to ultra high pressure liquid chromatography (UHPLC)-MS or other fast LC-MS techniques (core-shell particles, high-temperature LC and monolithic columns) requiring fast MS analyzers (typically time-of-flight (TOF)-based systems); (b) the use of supercritical fluid chromatography (SFC) for fast and “green” separations, with reduction in solvents consumption; (c) the use of multidimensional liquid chromatography techniques (MDLC) for complex samples, and other dimension also in MS, such as ion mobility spectrometry (IMS)-MS, the coupling of two or more mass analyzer (tandem MS); (d) the shift from low-resolution to (ultra)high-resolution MS to allow accurate mass measurements. Each of these techniques will be described in detail in the following chapters, illustrating selected applications developed for the analysis of lipid and lipid-like molecules, as well as other bioactive compounds.