TY - JOUR
T1 - Unified-Hydrophilic-Interaction/Anion-Exchange Liquid Chromatography Mass Spectrometry (Unified-HILIC/AEX/MS)
T2 - A Single-Run Method for Comprehensive and Simultaneous Analysis of Polar Metabolome
AU - Nakatani, Kohta
AU - Izumi, Yoshihiro
AU - Takahashi, Masatomo
AU - Bamba, Takeshi
N1 - Funding Information:
We thank ChemAxon (Budapest, Hungary) for using ChemAxon software. This study was supported by JST-Mirai (JPMJMI20G1 for Y.I.) of the Japan Science and Technology Agency (JST); JST-Moonshot (JPMJMS2011-62 for Y.I.); JST-CREST (JPMJCR22N5 for Y.I. and JPMJCR15G4 for T.B.), JST A-STEP (JPMJTR204J for T.B.); AMED-CREST (JP22gm1010010 for T.B) from Japan Agency for Medical Research and Development (AMED); a project (JPNP20011 for T.B.) subsidized by the New Energy and Industrial Technology Development Organization (NEDO); and KAKENHI (JP21K14472 for K.N., JP22H01883 for Y.I., JP22K18924 for Y.I., JP22H05185 for Y.I., JP20K15101 for M.T., JP17H06304 for T.B., and JP18H01800 for T.B.) from Japan Society for the Promotion of Science (JSPS).
Publisher Copyright:
© 2022 American Chemical Society.
PY - 2022/12/6
Y1 - 2022/12/6
N2 - One of the technical challenges in the field of metabolomics is the development of a single-run method to detect the full complement of polar metabolites in biological samples. However, an ideal method to meet this demand has not yet been developed. Herein, we proposed a simple methodology that enables the comprehensive and simultaneous analysis of polar metabolites using unified-hydrophilic-interaction/anion-exchange liquid chromatography mass spectrometry (unified-HILIC/AEX/MS) with a polymer-based mixed amines column composed of methacrylate-based polymer particles with primary, secondary, tertiary, and quaternary amines as functional groups. The optimized unified-HILIC/AEX/MS method is composed of two consecutive chromatographic separations, HILIC-dominant separation for cationic, uncharged, and zwitterionic polar metabolites [retention times (RTs) = 0-12.8 min] and AEX-dominant separation for polar anionic metabolites (RTs = 12.8-26.5 min), by varying the ratio of acetonitrile to 40 mM ammonium bicarbonate solution (pH 9.8). A total of 400 polar metabolites were analyzed simultaneously through a combination of highly efficient separation using unified-HILIC/AEX and remarkably sensitive detection using multiple reaction monitoring-based triple quadrupole mass spectrometry (unified-HILIC/AEX/MS/MS). A nontargeted metabolomic approach using unified-HILIC/AEX high-resolution mass spectrometry (unified-HILIC/AEX/HRMS) also provided more comprehensive information on polar metabolites (3242 metabolic features) in HeLa cell extracts than the conventional HILIC/HRMS method (2068 metabolic features). Our established unified-HILIC/AEX/MS/MS and unified-HILIC/AEX/HRMS methods have several advantages over conventional techniques, including polar metabolome coverage, throughput, and accurate quantitative performance, and represent potentially useful tools for in-depth studies on metabolism and biomarker discovery.
AB - One of the technical challenges in the field of metabolomics is the development of a single-run method to detect the full complement of polar metabolites in biological samples. However, an ideal method to meet this demand has not yet been developed. Herein, we proposed a simple methodology that enables the comprehensive and simultaneous analysis of polar metabolites using unified-hydrophilic-interaction/anion-exchange liquid chromatography mass spectrometry (unified-HILIC/AEX/MS) with a polymer-based mixed amines column composed of methacrylate-based polymer particles with primary, secondary, tertiary, and quaternary amines as functional groups. The optimized unified-HILIC/AEX/MS method is composed of two consecutive chromatographic separations, HILIC-dominant separation for cationic, uncharged, and zwitterionic polar metabolites [retention times (RTs) = 0-12.8 min] and AEX-dominant separation for polar anionic metabolites (RTs = 12.8-26.5 min), by varying the ratio of acetonitrile to 40 mM ammonium bicarbonate solution (pH 9.8). A total of 400 polar metabolites were analyzed simultaneously through a combination of highly efficient separation using unified-HILIC/AEX and remarkably sensitive detection using multiple reaction monitoring-based triple quadrupole mass spectrometry (unified-HILIC/AEX/MS/MS). A nontargeted metabolomic approach using unified-HILIC/AEX high-resolution mass spectrometry (unified-HILIC/AEX/HRMS) also provided more comprehensive information on polar metabolites (3242 metabolic features) in HeLa cell extracts than the conventional HILIC/HRMS method (2068 metabolic features). Our established unified-HILIC/AEX/MS/MS and unified-HILIC/AEX/HRMS methods have several advantages over conventional techniques, including polar metabolome coverage, throughput, and accurate quantitative performance, and represent potentially useful tools for in-depth studies on metabolism and biomarker discovery.
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U2 - 10.1021/acs.analchem.2c03986
DO - 10.1021/acs.analchem.2c03986
M3 - Article
AN - SCOPUS:85143063273
VL - 94
SP - 16877
EP - 16886
JO - Analytical Chemistry
JF - Analytical Chemistry
SN - 0003-2700
IS - 48
ER -