title: A novel liquid chromatography tandem mass spectrometry method for simultaneous determination of branched-chain amino acids and branched-chain α-keto acids in human plasma.
abstract: Branched-chain amino acids (BCAAs) and branched-chain α-keto acids (BCKAs) play significant biological roles as they are involved in protein and neurotransmitter synthesis as well as energy metabolism pathways. To routinely and accurately study the dynamics of BCAAs and BCKAs in human diseases, e.g. cerebral infarction, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated. The plasma samples were deproteinized with acetonitrile, and then separated on a reversed phase C18 column with a mobile phase of 0.1 % formic acid (solvent A)-methanol (solvent B) using gradient elution. The detection of BCAAs and BCKAs was conducted in multiple reaction monitoring with positive/negative electrospray ionization switching mode. Biologically relevant isomers such as leucine and isoleucine were individually quantified by combining chromatographic separation and fragmentation. Good linearity (R (2) > 0.99) was obtained for all six analytes with the limits of detection from 0.1 to 0.2 µg/mL. The intra-day and inter-day accuracy ranged from 93.7 to 108.4 % and the relative standard deviation (RSD) did not exceed 15.0 %. The recovery was more than 80 % with RSD less than 14.0 %. The main improvements compared to related, state-of-the-art methods included enhanced sensitivity, enhanced separation of isomers, and reduced complexity of sample processing. Finally, the validated method was applied to analyze the plasma samples of healthy volunteers and patients suffering cerebral infarction, and significant differences in the concentration levels of BCAAs and BCKAs were observed.
authors.affiliation: Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Ministry of Education, Tongjiaxiang No. 24, Nanjing, 210009, China.
authors.forename: Ruiting
authors.initials: R
authors.lastname: Li
authors.affiliation: Department of Neurology, the Second Affiliated Hospital, Harbin Medical University, Harbin, China.
authors.forename: Peifang
authors.initials: P
authors.lastname: Liu
authors.affiliation: Department of Neurology, the Second Affiliated Hospital, Harbin Medical University, Harbin, China.
authors.forename: Peijia
authors.initials: P
authors.lastname: Liu
authors.affiliation: Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Ministry of Education, Tongjiaxiang No. 24, Nanjing, 210009, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Tian
authors.affiliation: Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Ministry of Education, Tongjiaxiang No. 24, Nanjing, 210009, China.
authors.forename: Yunfei
authors.initials: Y
authors.lastname: Hua
authors.affiliation: Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Ministry of Education, Tongjiaxiang No. 24, Nanjing, 210009, China.
authors.forename: Yiqiao
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, China.
authors.forename: Hua
authors.initials: H
authors.lastname: He
authors.affiliation: Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Ministry of Education, Tongjiaxiang No. 24, Nanjing, 210009, China.
authors.forename: Jiaqing
authors.initials: J
authors.lastname: Chen
authors.affiliation: Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Ministry of Education, Tongjiaxiang No. 24, Nanjing, 210009, China. zunjianzhangcpu@hotmail.com.
authors.forename: Zunjian
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Ministry of Education, Tongjiaxiang No. 24, Nanjing, 210009, China. huangyin@cpu.edu.cn.
authors.forename: Yin
authors.initials: Y
authors.lastname: Huang
mesh_terms: D000597:Amino Acids, Branched-Chain / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007651:Keto Acids / Q000097:blood*; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D000597:Amino Acids, Branched-Chain; D007651:Keto Acids
keywords: Branched-chain amino acids; Branched-chain α-keto acids; Cerebral infarction; Human plasma; Isomer; LC–MS/MS