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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

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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.

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References

  • Almeida CC, Alvares TS, Costa MP, Conte-Junior CA (2016) Protein and amino acid profiles of different whey protein supplements. J Diet Suppl 13:313–323. doi:10.3109/19390211.2015.1036187

    Article  CAS  PubMed  Google Scholar 

  • Bouri M, Salghi R, Zougagh M, Ríos A (2013) Capillary electrophoresis coupled to evaporative light scattering detection for direct determination of underivatized amino acids: application to tea samples using carboxyled single-walled carbon nanotubes for sample preparation. Electrophoresis 34:2623–2631. doi:10.1002/elps.201300145

    Article  CAS  PubMed  Google Scholar 

  • Bruce SJ, Tavazzi I, Parisod V et al (2009) Investigation of human blood plasma sample preparation for performing metabolomics using ultrahigh performance liquid chromatography/mass spectrometry. Anal Chem 81:3285–3296. doi:10.1021/ac8024569

    Article  CAS  PubMed  Google Scholar 

  • Buescher JM, Moco S, Sauer U, Zamboni N (2010) Ultrahigh performance liquid chromatography–tandem mass spectrometry method for fast and robust quantification of anionic and aromatic metabolites. Anal Chem 82:4403–4412. doi:10.1021/ac100101d

    Article  CAS  PubMed  Google Scholar 

  • Christou C, Gika HG, Raikos N, Theodoridis G (2014) GC-MS analysis of organic acids in human urine in clinical settings: a study of derivatization and other analytical parameters. J Chromatogr B Analyt Technol Biomed Life Sci 964:195–201. doi:10.1016/j.jchromb.2013.12.038

    Article  CAS  PubMed  Google Scholar 

  • Del Campo G, Zuriarrain J, Zuriarrain A, Berregi I (2016) Quantitative determination of carboxylic acids, amino acids, carbohydrates, ethanol and hydroxymethylfurfural in honey by (1)H NMR. Food Chem 196:1031–1039. doi:10.1016/j.foodchem.2015.10.036

    Article  PubMed  Google Scholar 

  • Dettmer K, Stevens AP, Fagerer SR et al (2012) Amino acid analysis in physiological samples by GC-MS with propyl chloroformate derivatization and iTRAQ-LC–MS/MS. Methods Mol Biol 828:165–181. doi:10.1007/978-1-61779-445-2_15

    Article  CAS  PubMed  Google Scholar 

  • Domingues DS, Crevelin EJ, de Moraes LAB et al (2015) Simultaneous determination of amino acids and neurotransmitters in plasma samples from schizophrenic patients by hydrophilic interaction liquid chromatography with tandem mass spectrometry. J Sep Sci 38:780–787. doi:10.1002/jssc.201400943

    Article  CAS  PubMed  Google Scholar 

  • Ehling S, Reddy TM (2015) Direct analysis of leucine and Its metabolites β-hydroxy-β-methylbutyric Acid, α-ketoisocaproic acid, and α-hydroxyisocaproic acid in human breast milk by liquid chromatography–mass spectrometry. J Agric Food Chem 63:7567–7573. doi:10.1021/acs.jafc.5b02563

    Article  CAS  PubMed  Google Scholar 

  • Fernstrom JD (2005) Branched-chain amino acids and brain function. J Nutr 135:1539S–1546S

    CAS  PubMed  Google Scholar 

  • Ghosh S, Sengupta A, Chandra K (2015) Quantitative metabolic profiling of NMR spectral signatures of branched chain amino acids in blood serum. Amino Acids 47:2229–2236. doi:10.1007/s00726-015-1994-1

    Article  CAS  PubMed  Google Scholar 

  • Giesbertz P, Daniel H (2016) Branched-chain amino acids as biomarkers in diabetes. Curr Opin Clin Nutr Metab Care 19:48–54. doi:10.1097/MCO.0000000000000235

    Article  CAS  PubMed  Google Scholar 

  • González O, Blanco ME, Iriarte G et al (2014) Bioanalytical chromatographic method validation according to current regulations, with a special focus on the non-well defined parameters limit of quantification, robustness and matrix effect. J Chromatogr A 1353:10–27. doi:10.1016/j.chroma.2014.03.077

    Article  PubMed  Google Scholar 

  • Harper AE, Miller RH, Block KP (1984) Branched-chain amino acid metabolism. Annu Rev Nutr 4:409–454. doi:10.1146/annurev.nu.04.070184.002205

    Article  CAS  PubMed  Google Scholar 

  • Harris RA, Joshi M, Jeoung NH, Obayashi M (2005) Overview of the molecular and biochemical basis of branched-chain amino acid catabolism. J Nutr 135:1527S–1530S

    CAS  PubMed  Google Scholar 

  • Henriquez H, El Din A, Ozand PT et al (1994) Emergency presentations of patients with methylmalonic acidemia, propionic acidemia and branched chain amino acidemia (MSUD). Brain and Development 16:86–93. doi:10.1016/0387-7604(94)90101-5

    Article  PubMed  Google Scholar 

  • How ZT, Busetti F, Linge KL et al (2014) Analysis of free amino acids in natural waters by liquid chromatography–tandem mass spectrometry. J Chromatogr A 1370:135–146. doi:10.1016/j.chroma.2014.10.040

    Article  CAS  PubMed  Google Scholar 

  • Kand’ár R, Záková P, Jirosová J, Sladká M (2009) Determination of branched chain amino acids, methionine, phenylalanine, tyrosine and alpha-keto acids in plasma and dried blood samples using HPLC with fluorescence detection. Clin Chem Lab Med 47:565–572. doi:10.1515/CCLM.2009.123

    PubMed  Google Scholar 

  • Kato S, Kito Y, Hemmi H, Yoshimura T (2011) Simultaneous determination of d-amino acids by the coupling method of d-amino acid oxidase with high-performance liquid chromatography. J Chromatogr B Analyt Technol Biomed Life Sci 879:3190–3195. doi:10.1016/j.jchromb.2010.12.005

    Article  CAS  PubMed  Google Scholar 

  • Krumpochova P, Bruyneel B, Molenaar D et al (2015) Amino acid analysis using chromatography–mass spectrometry: an inter platform comparison study. J Pharm Biomed Anal 114:398–407. doi:10.1016/j.jpba.2015.06.001

    Article  CAS  PubMed  Google Scholar 

  • Liu J, Liu M, Li X et al (2014) Development of ultrasonic-assisted closed in-syringe extraction and derivatization for the determination of labile abietic acid and dehydroabietic acid in cosmetics. J Chromatogr A 1371:20–29. doi:10.1016/j.chroma.2014.10.059

    Article  CAS  PubMed  Google Scholar 

  • Miller JH IV, Poston PA, Karnes HT (2012) A quantitative method for acylcarnitines and amino acids using high resolution chromatography and tandem mass spectrometry in newborn screening dried blood spot analysis. J Chromatogr B Analyt Technol Biomed Life Sci 903:142–149. doi:10.1016/j.jchromb.2012.07.008

    Article  CAS  PubMed  Google Scholar 

  • Nguyen D-T, Lee G, Paik M-J (2013) Keto acid profiling analysis as ethoxime/tert-butyldimethylsilyl derivatives by gas chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 913–914:48–54. doi:10.1016/j.jchromb.2012.11.021

    Article  PubMed  Google Scholar 

  • Noguchi K, Mizukoshi T, Miyano H, Yamada N (2014) Development of a new LC–MS/MS method for the quantification of keto acids. Chromatography 35:117–123. doi:10.15583/jpchrom.2014.017

    Article  Google Scholar 

  • Önal A, Tekkeli SEK, Önal C (2013) A review of the liquid chromatographic methods for the determination of biogenic amines in foods. Food Chem 138:509–515. doi:10.1016/j.foodchem.2012.10.056

    Article  PubMed  Google Scholar 

  • Qi W, Guan Q, Sun T et al (2015) Improving detection sensitivity of amino acids in thyroid tissues by using phthalic acid as a mobile phase additive in hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometry. Anal Chim Acta 870:75–82. doi:10.1016/j.aca.2015.02.048

    Article  CAS  PubMed  Google Scholar 

  • Qureshi GA (1987) High-performance liquid chromatographic methods with fluorescence detection for the determination of branched-chain amino acids and their alpha-keto analogues in plasma samples of healthy subjects and uraemic patients. J Chromatogr 400:91–99

    Article  CAS  PubMed  Google Scholar 

  • Ribeiro CA, Sgaravatti AM, Rosa RB et al (2008) Inhibition of brain energy metabolism by the branched-chain amino acids accumulating in maple syrup urine disease. Neurochem Res 33:114–124. doi:10.1007/s11064-007-9423-9

    Article  CAS  PubMed  Google Scholar 

  • Ruiz-Angel MJ, García-Alvarez-Coque MC, Berthod A, Carda-Broch S (2014) Are analysts doing method validation in liquid chromatography? J Chromatogr A 1353:2–9. doi:10.1016/j.chroma.2014.05.052

    Article  CAS  PubMed  Google Scholar 

  • Shimomura Y, Honda T, Shiraki M et al (2006) Branched-chain amino acid catabolism in exercise and liver disease. J Nutr 136:250S–253S

    CAS  PubMed  Google Scholar 

  • Takach E, O’Shea T, Liu H (2014) High-throughput quantitation of amino acids in rat and mouse biological matrices using stable isotope labeling and UPLC–MS/MS analysis. J Chromatogr B Analyt Technol Biomed Life Sci 964:180–190. doi:10.1016/j.jchromb.2014.04.043

    Article  CAS  PubMed  Google Scholar 

  • Tang D-Q, Zou L, Yin X-X, Ong CN (2014) HILIC-MS for metabolomics: an attractive and complementary approach to RPLC–MS. Mass Spectrom Rev. doi:10.1002/mas.21445

    PubMed  Google Scholar 

  • Tom A, Nair KS (2006) Assessment of branched-chain amino acid status and potential for biomarkers. J Nutr 136:324S–330S

    CAS  PubMed  Google Scholar 

  • Wang C, Zhang W, Song F et al (2011) A simple method for the analysis by MS/MS of underivatized amino acids on dry blood spots from newborn screening. Amino Acids 42:1889–1895. doi:10.1007/s00726-011-0910-6

    Article  PubMed  Google Scholar 

  • Ziegler J, Abel S (2014) Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization. Amino Acids 46:2799–2808. doi:10.1007/s00726-014-1837-5

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This study was financially supported by the Open Project Program of Key Laboratory of Myocardial Ischemia (Harbin Medical University), Ministry of Education (No. KF201414); the Open Project Program of Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education (No. MKLDP2013QN04); the Innovative Scientific Research Team Fund of Jiangsu Province; and the Priority Academic Program Development of Jiangsu Higher Education Institutions. The authors would like to thank the patients for their participation in our project.

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Authors and Affiliations

  1. Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University, Ministry of Education, Tongjiaxiang No. 24, Nanjing, 210009, China

    Ruiting Li, Yuan Tian, Yunfei Hua, Yiqiao Gao, Jiaqing Chen, Zunjian Zhang & Yin Huang

  2. Department of Neurology, the Second Affiliated Hospital, Harbin Medical University, Harbin, China

    Peifang Liu & Peijia Liu

  3. Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, China

    Hua He

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  1. Ruiting Li
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  2. Peifang Liu
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  4. Yuan Tian
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  5. Yunfei Hua
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  6. Yiqiao Gao
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  7. Hua He
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  9. Zunjian Zhang
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  10. Yin Huang
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Correspondence to Zunjian Zhang or Yin Huang.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Handling editor: D. Tsikas.

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Li, R., Liu, P., Liu, P. et al. 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. Amino Acids 48, 1523–1532 (2016). https://doi.org/10.1007/s00726-016-2212-5

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  • DOI: https://doi.org/10.1007/s00726-016-2212-5

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