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Untargeted analysis performed using full-scan mass spectrometry (MS) coupled with liquid chromatography (LC) is commonly used in metabolomics. Although they are commonly employed, full-scan MS methods such as quadrupole-time-of-flight (Q-TOF) MS have been restricted by various factors including their limited linear range and complicated data processing. LC coupled with triple quadrupole (QQQ) MS operated in the multiple reaction monitoring (MRM) mode is the gold standard for metabolite quantification; however, only known metabolites are generally quantified, limiting its applications in metabolomic analysis. In this study, a pseudotargeted approach was proposed to perform serum metabolomic analysis using an ultra high-performance liquid chromatography (UHPLC)/QQQ MS system operated in the MRM mode, for which the MRM ion pairs were acquired from the serum samples through untargeted tandem MS using UHPLC/Q-TOF MS. The UHPLC/QQQ MRM MS-based pseudotargeted method displayed better repeatability and wider linear range than the traditional UHPLC/Q-TOF MS-based untargeted metabolomics method, and no complicated peak alignment was required. The developed method was applied to discover serum biomarkers for patients with hepatocellular carcinoma (HCC). Patients with HCC had decreased lysophosphatidylcholine, increased long-chain and decreased medium-chain acylcarnitines, and increased aromatic and decreased branched-chain amino acid levels compared to healthy controls. The novelty of this work is that it provides an approach to acquire MRM ion pairs from real samples, is not limited to metabolite standards, and it provides a foundation to achieve pseudotargeted metabolomic analysis on the widely used LC/QQQ MS platform. [ABSTRACT FROM AUTHOR]