title: Evaluation of a novel system for analyzing hydrophilic blood metabolites.
abstract: Metabolomics has recently been developed, and there have been a considerable number of metabolomics-based biomarker studies in the medical research field. Therefore, as a first step toward the practical use of metabolite biomarkers, a simple and quick sample preparation method involving metabolite extraction, metabolite measurement, and data analysis needs to be developed. In this study, we evaluated whether the use of simpler metabolite extraction methods would facilitate the stable analysis of hydrophilic blood metabolites during liquid chromatography/triple quadrupole mass spectrometry (LC/QqQMS)-based metabolome analysis. As a result, the anion and cation metabolites in plasma were stably analyzed via a methanol-based extraction procedure followed by ultrafiltration, and it was also confirmed that a lyophilization step was not necessary. When extraction was performed without a lyophilization step, approximately >50% and >80% of the detected metabolites had relative standard deviation values of <20% during LC/QqQMS-based anion and cation analyses, respectively. In addition, the plasma levels of l-valine, l-leucine, l-isoleucine, l-tyrosine, and l-phenylalanine were quantitatively measured using the corresponding stable isotopes; the SCLAM-2000, a fully automatic pre-treatment system for LC/MS that can be connected online to an LC/MS device; and an extraction procedure based on the simple procedure that we developed. Our findings suggest that simpler pretreatment procedures can be employed during LC/QqQMS-based metabolomics and might aid searches for metabolite biomarker candidates, the validation of metabolite biomarker candidates, and the practical use of metabolite biomarkers.
authors.affiliation: Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
authors.forename: Shin
authors.initials: S
authors.lastname: Nishiumi
authors.affiliation: Global Application Development Center, Analytical & Measuring Instruments Division, Shimadzu Corporation, 1 Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto 604-8511, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Shima
authors.affiliation: Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Azuma
authors.affiliation: Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan; Division of Metabolomics Research, Department of Internal Related, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan; AMED-CREST, Japan Agency for Medical Research and Development, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan. Electronic address: myoshida@med.kobe-u.ac.jp.
authors.forename: Masaru
authors.initials: M
authors.lastname: Yoshida
mesh_terms: D000596:Amino Acids / Q000097:blood* / Q000737:chemistry*; D015415:Biomarkers / Q000097:blood / Q000737:chemistry; D002853:Chromatography, Liquid; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D055432:Metabolomics / Q000379:methods*
chemical_list: D000596:Amino Acids; D015415:Biomarkers
keywords: Aromatic amino acid; Branched-chain amino acid; Fischer ratio; Liquid chromatography/triple quadrupole mass spectrometry; Metabolite; Metabolomics