Comprehensive Analytical Method for the Determination of Hydrophilic Metabolites by High-Performance Liquid Chromatography and Mass Spectrometry

Abstract

A method for the comprehensive analysis of hydrophilic metabolites, based on a combination of high-performance liquid chromatography and mass spectrometry, is described. We evaluated three types of stationary phases to achieve the separation of highly hydrophilic metabolites. Good chromatographic retention and separation of these metabolites were achieved on a pentafluorophenylpropyl-bonded silica column with gradient elution, using 0.1% aqueous formic acid and acetonitrile as the mobile phase. The optimized conditions allowed the comprehensive determination of the standard 49 kinds of amino acids, 6 kinds of amines, 45 kinds of organic acids, 18 kinds of nucleic bases, 5 kinds of nucleosides, and 14 kinds of nucleotides, and then the linearity, dynamic range, detection limit, and precision of the retention time and the peak area were validated. We applied this method for the targeted analysis of the components in soy sauce. The results from the quantitative determination of amino acids were compared to those obtained with an amino acid analyzer, and the accuracy was in the range between 85 and 119%. The accuracy of other detected components was confirmed to be 105-133% by the recovery rate after the addition of standard compounds. We also applied the method for the nontargeted metabolic profiling of the components in several kinds of soy sauces with the principal component analysis. They were classified by the manufacturing methods, and the components that corresponded to the differences were identified. This method could be useful for the targeted analysis of hydrophilic metabolites as well as their nontargeted metabolic profiling.

Technical Challenges in Mass Spectrometry-Based Metabolomics

“…is is also because there has been no de nite and versatile inlet method that is able to simultaneously separate a wide variety of metabolites. In the case of LC/MS metabolomics, there are at least 5 groups of LC conditions, which include reverse phase analysis using octadecylsilyl (ODS) column for relatively hydrophobic metabolites, 45) normal phase analysis using columns for hydrophilic interaction chromatography (HILIC) of hydrophilic metabolites, [46][47][48] reverse phase analysis using pentafluorophenylpropyl stationary phase column for hydrophilic metabolites, 49) ion-paring methods using ODS column for anionic metabolites, 50) and lipidomics methods using ODS column for lipids. Recently, a comparison of the chromatographic performance of a traditional ODS column with various HILIC and mixed-mode columns showed that ODS and zwitterionic HILIC columns are the best combination for wide metabolite coverage.…”

Clinical Implementation of Metabolomics

“…This was a weakness of LC-MS analysis, and LC-MS was limited to the analysis of hydrophobic metabolites such as lipids. However, the development of column technology enabled the retention and separation of hydrophilic metabolites (Alpert, 1990;Yoshida et al, 2007). This technology has been applied for the research of drug metabolites (Plumb et al, 2003), galactosamine toxicity (Spagou et al, 2011), and renal cell carcinoma diagnosis, staging, and biomarker discovery (Lin et al, 2011a).…”

Amino acid analysis of sub-picomolar amounts of proteins by precolumn fluorescence derivatization with 6-aminoquinolyl-<i>N</i>-hydroxysuccinimidyl carbamate

“…T h e r e a r e s o m e a p p r o a c h e s w i t h o u t a n y derivatization of amino acids for MS detection coupled to capillary electrophoresis (11) or LC using a pentafluorophenylpropyl-bonded silica column (12) or a porous graphitic carbon column (13). Such highly sensitive AAA described above can complement MS-based proteomics of low abundance proteins.…”