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Development and validation of a rapid, selective, and sensitive LC–MS/MS method for simultaneous determination of d- and l-amino acids in human serum: application to the study of hepatocellular carcinoma

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Abstract

A validated liquid chromatography–tandem mass spectrometry method was developed for the simultaneous determination of d- and l-amino acids in human serum. Under the optimum conditions, except for dl-proline, l-glutamine, and d-lysine, the enantioseparation of the other 19 enantiomeric pairs of proteinogenic amino acids and nonchiral glycine was achieved with a CROWNPAK CR-I(+) chiral column within 13 min. The lower limits of quantitation for l-amino acids (including glycine) and d-amino acids were 5–56.25 μM and 0.625–500 nM, respectively, in human serum. The intraday precision and interday precision for all the analytes were less than 15%, and the accuracy ranged from −12.84% to 12.37% at three quality control levels. The proposed method, exhibiting high rapidity, enantioresolution, and sensitivity, was successfully applied to the quantification of d- and l-amino acid levels in serum from hepatocellular carcinoma patients and healthy individuals. The serum concentrations of l-arginine, l-isoleucine, l-aspartate, l-tryptophan, l-alanine, l-methionine, l-serine, glycine, l-valine, l-leucine, l-phenylalanine, l-threonine, d-isoleucine, d-alanine, d-glutamate, d-glutamine, d-methionine, and d-threonine were significantly reduced in the hepatocellular carcinoma patients compared with the healthy individuals (P < 0.01). d-Glutamate and d-glutamine were identified as the most downregulated serum markers (fold change greater than 1.5), which deserves further attention in hepatocellular carcinoma research.

Simultaneous determination of d- and l-amino acids in human serum from hepatocellular carcinoma patients and healthy individuals. AA amino acid, HCC hepatocellular carcinoma, LC liquid chromatography, MS/MS tandem mass spectrometry, NC normal control, TIC total ion chromatogram

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Acknowledgements

We gratefully thank the National Natural Science Foundation of China (no. 81101027), the Innovation Program of Shanghai Municipal Education Commission (no. 14YZ030), and the Shanghai Young Eastern Scholar Program (QD2015009) for financial support.

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  1. Minlu Han and Mengyu Xie contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, No. 280 South Chongqing Road, Shanghai, 200025, China

    Minlu Han, Mengyu Xie, Daoyi Yuan & Ying Xie

  2. Department of Hepatobiliary Surgery, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, 200438, China

    Jun Han & Tian Yang

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  1. Minlu Han
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Correspondence to Ying Xie.

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This study was approved by the ethics committees of Eastern Hepatobiliary Surgery Hospital and Changhai Hospital and was conducted in accordance with the guidelines of the World Medical Association’s Declaration of Helsinki. Informed consent was obtained from the individual participants who provided the blood samples.

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Han, M., Xie, M., Han, J. et al. Development and validation of a rapid, selective, and sensitive LC–MS/MS method for simultaneous determination of d- and l-amino acids in human serum: application to the study of hepatocellular carcinoma. Anal Bioanal Chem 410, 2517–2531 (2018). https://doi.org/10.1007/s00216-018-0883-3

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