These elution conditions may, however, affect MS-based detection by reducing the ionization yield of the target analyte [63,68]. In general, highly polar compounds can be separated by (i) hydrophilic interaction chromatography (HILIC), (ii) the addition of ion-pairing agents to the mobile phase, and (iii) the incorporation of ion ligands within conventional RP surfaces to enable mixed mode separation [33]. In particular, HILIC, which was developed by Alpert for the separation of highly polar compounds in 1990 [69], is a powerful separation technique. HILIC-MS

was employed to separate and quantify highly polar compounds in #BIBW2992 keyword# biological samples [70,71,72]. In HILIC, retention increases with increasing polarity of the stationary phase and solutes and with decreasing polarity of the organic solvent systems used for elution; this contrasts with the trend observed with RPLC. Recently, HILIC was coupled to MS and used in metabolomics studies [73]. Analytical methods for biological samples were developed [74,75,76,77], the focus of Inhibitors,research,lifescience,medical which was on metabolic pathways. Metabolic profiling offers important information for interpreting the efficacy Inhibitors,research,lifescience,medical outcome, explaining the toxicity of lead compounds, and rationalizing the toxicity of specific drugs during drug discovery or development [78]. HILIC clearly provides different selectivity from RPLC for the separation of highly polar

compounds [73,79]. Therefore, HILIC is useful for determining polar metabolic compounds. LC-MS applications are summarized in Table 2. Table 2 Summary of LC-MS Inhibitors,research,lifescience,medical applications in metabolomics. 4. Applications 4.1. Amino Acids Amino acids are precursors for the synthesis of proteins, other nitrogenous substances, glucose, and fatty acids [90,91]. Amino acids play a major role in energy metabolism, neurotransmission, and lipid transport, and are important in disease diagnostics, and in elucidating nutritional influences Inhibitors,research,lifescience,medical on physiology [92,93]. Amino acids not only act as building blocks of proteins but also serve as key regulators of metabolic

pathways in cells. However, the mechanisms responsible for the effects of amino acids are largely unknown. Metabolomics studies are very difficult to perform because of the wide variability of biological fluids (mainly urine) in association with different confounding factors, such as gender, age, time of day, state of health, lifestyle, diet, and phenotype [94]. Therefore, the 3-mercaptopyruvate sulfurtransferase integration of various omics technologies and bioinformatics with conventional techniques is expected to provide comprehensive information about amino acid metabolism and nutrition in organisms. Waldhier et al. applied GC×GC-TOFMS to the separation of amino acid enantiomers after derivatization with methyl chloroformate [95], and were able to successfully separate 10 amino acid enantiomers from serum and urine matrix. On the other hand, Williams et al.