title: A novel quantification method for analysis of twenty natural amino acids in human serum based on N-phosphorylation labeling using reversed-phase liquid chromatography-tandem mass spectrometry.
abstract: A novel method based on the strategy of N-phosphorylation labeling is described for quantification of twenty natural amino acids in human serum by reversed-phase liquid chromatography-electrospray tandem mass spectrometry (RP-LC/ESI-MS). The derivatization reaction was easily performed in one-pot reaction under mild conditions within 30min. The reaction mixture was then evaporated to dryness, redissolved, desalted by C18 SPE. The twenty N-phosphoryl amino acids were separated on an RP-C18 column within 20min by isocratic elution (0.1% formic acid-acetonitrile, v/v 7:3). At the same time, multiple reaction monitoring (MRM) MS enabled quantitation of twenty natural amino with the LOD of 0.0005-0.15μM and LOQ of 0.0020-0.5μM in human serum. The linear range was from 0.025 to 25μM (except Cys and Trp) with R>0.99. The recovery range was determined to be 85.5-117.4% with the relative standard deviation (RSD) in the range of 1.3-13.9%. All twenty amino acids were successfully detected in human serum samples with the concentration from 5.7 to 577.9μM, which indicates potential of the developed method for determination of amino acids in complex biological samples, hence for screening of amino acid metabolite related diseases.
authors: Department of Chemistry, Tsinghua University, Beijing 100084, China; Key Laboratory of Metabolomics at Shenzhen, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China; State Key Laboratory Breeding Base-Shenzhen Key Laboratory of Chemical Biology, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China.
authors: Xiaowu
authors: X
authors: Chen
authors: Key Laboratory of Metabolomics at Shenzhen, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China; State Key Laboratory Breeding Base-Shenzhen Key Laboratory of Chemical Biology, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China.
authors: Dan
authors: D
authors: Gao
authors: Key Laboratory of Metabolomics at Shenzhen, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China; State Key Laboratory Breeding Base-Shenzhen Key Laboratory of Chemical Biology, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China.
authors: Feng
authors: F
authors: Liu
authors: School of Pharmaceutical Sciences, Xiamen University, Xiamen 361102, China.
authors: Xiang
authors: X
authors: Gao
authors: Key Laboratory of Metabolomics at Shenzhen, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China; State Key Laboratory Breeding Base-Shenzhen Key Laboratory of Chemical Biology, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China.
authors: Shujuan
authors: S
authors: Wang
authors: State Key Laboratory Breeding Base-Shenzhen Key Laboratory of Chemical Biology, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China.
authors: Yufen
authors: Y
authors: Zhao
authors: Key Laboratory of Metabolomics at Shenzhen, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China; State Key Laboratory Breeding Base-Shenzhen Key Laboratory of Chemical Biology, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China. Electronic address: liuhx@sz.tsinghua.edu.cn.
authors: Hongxia
authors: H
authors: Liu
authors: State Key Laboratory Breeding Base-Shenzhen Key Laboratory of Chemical Biology, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China; School of Medicine, Tsinghua University, Beijing 100084, China. Electronic address: jiangyy@sz.tsinghua.edu.cn.
authors: Yuyang
authors: Y
authors: Jiang
mesh_terms: D000596:Amino Acids / Q000097:blood*; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D010766:Phosphorylation; D012016:Reference Values; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000596:Amino Acids
keywords: Amino acids; Human serum; LC–MS/MS; N-phosphorylation labeling; Reversed-phase liquid chromatography