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 SCLAM2000, 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. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.