Amino Acids

, Volume 48, Issue 7, pp 1731–1735 | Cite as

Rapid quantitative method for the detection of phenylalanine and tyrosine in human plasma using pillar array columns and gradient elution

  • Yanting Song
  • Katsuya Takatsuki
  • Tetsushi Sekiguchi
  • Takashi Funatsu
  • Shuichi Shoji
  • Makoto TsunodaEmail author
Short Communication


This study reports a fast and quantitative determination method for phenylalanine (Phe) and tyrosine (Tyr) in human plasma using on-chip pressure-driven liquid chromatography. A pillar array column with low-dispersion turns and a gradient elution system was used. The separation of fluorescent derivatives of Phe, Tyr, and other hydrophobic amino acids was successfully performed within 140 s. Under the optimized conditions, Phe and Tyr in human plasma were quantified. The developed method is promising for rapid diagnosis in the clinical field.


Microfluidics 4-Fluoro-7-nitro-2,1,3-benzoxadiazole Fluorescence Quantitative determination Amino acids 



This research is supported by the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C) grant number 26460033 and the Center of Innovation Program from Japan Science and Technology Agency, JST.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants performed by any of the authors.


  1. Aoyama C, Saeki A, Noguchi M, Shirasaki Y, Shoji S, Funatsu T, Mizuno J, Tsunoda M (2010) Use of folded micromachined pillar array column with low-dispersion turns for pressure-driven liquid chromatography. Anal Chem 82(4):1420–1426CrossRefPubMedGoogle Scholar
  2. Biermann M, Bardl B, Vollstaedt S, Linnemann J, Knuepfer U, Seidel G, Horn U (2013) Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra-high performance liquid chromatography approach. Amino Acids 44(4):1225–1231CrossRefPubMedPubMedCentralGoogle Scholar
  3. Broadley KJ (2010) The vascular effects of trace amines and amphetamines. Pharmacol Ther 125(3):363–375CrossRefPubMedGoogle Scholar
  4. Callewaert M, Desmet G, Ottevaere H, De Malsche W (2016) Detailed kinetic performance analysis of micromachined radially elongated pillar array columns for liquid chromatography. J Chromatogr A 1433:75–84CrossRefPubMedGoogle Scholar
  5. Contreras J, Alonso E, Fuentes LE (2015) HPLC for confirmatory diagnosis and biochemical monitoring of cuban patients with hyperphenylalaninemias. MEDICC Rev 17(1):23–28PubMedGoogle Scholar
  6. de Beeck JO, De Malsche W, Tezcan DS, De Moor P, Desmet G (2012) Impact of the limitations of state-of-the-art micro-fabrication processes on the performance of pillar array columns for liquid chromatography. J Chromatogr A 1239:35–48CrossRefGoogle Scholar
  7. De Malsche W, Eghbali H, Clicq D, Vangelooven J, Gardeniers H, Desmet G (2007) Pressure-driven reverse-phase liquid chromatography separations in ordered nonporous pillar array columns. Anal Chem 79(15):5915–5926CrossRefPubMedGoogle Scholar
  8. He B, Regnier F (1998) Microfabricated liquid chromatography columns based on collocated monolith support structures. J Pharm Biomed Anal 17(6–7):925–932CrossRefPubMedGoogle Scholar
  9. He B, Tait N, Regnier F (1998) Fabrication of nanocolumns for liquid chromatography. Anal Chem 70(18):3790–3797CrossRefPubMedGoogle Scholar
  10. Humphrey M, Nation J, Francis I, Boneh A (2011) Effect of tetrahydrobiopterin on Phe/Tyr ratios and variation in Phe levels in tetrahydrobiopterin responsive PKU patients. Mol Genet Metab 104(1–2):89–92CrossRefPubMedGoogle Scholar
  11. Kanamori T, Isokawa M, Funatsu T, Tsunoda M (2015) Development of analytical method for catechol compounds in mouse urine using hydrophilic interaction liquid chromatography with fluorescence detection. J Chromatogr B 985:142–148CrossRefGoogle Scholar
  12. Knox JH (2002) Band dispersion in chromatography–a universal expression for the contribution from the mobile zone. J Chromatogr A 960(1–2):7–18CrossRefPubMedGoogle Scholar
  13. Lindemann L, Hoener MC (2005) A renaissance in trace amines inspired by a novel GPCR family. Trends Pharmacol Sci 26(5):274–281CrossRefPubMedGoogle Scholar
  14. Neurauter G, Scholl-Buergi S, Haara A, Geisler S, Mayersbach P, Schennach H, Fuchs D (2013) Simultaneous measurement of phenylalanine and tyrosine by high performance liquid chromatography (HPLC) with fluorescence detection. Clin Biochem 46(18):1848–1851CrossRefPubMedGoogle Scholar
  15. Peat J, Garg U (2016) Determination of phenylalanine and tyrosine by high performance liquid chromatography-tandem mass spectrometry. Methods Mol Biol 1378:219–225CrossRefPubMedGoogle Scholar
  16. Pimentel FB, Alves RC, Costa ASG, Torres D, Almeida MF, Oliveira MBPP (2014) Phenylketonuria: protein content and amino acids profile of dishes for phenylketonuric patients. The relevance of phenylalanine. Food Chem 149:144–150CrossRefPubMedGoogle Scholar
  17. Sharman R, Sullivan K, Young R, McGill J (2010) A preliminary investigation of the role of the phenylalynine: tyrosine ratio in children with early and continuously treated phenylketonuria: toward identification of “safe” levels. Dev Neuropsychol 35(1):57–65CrossRefPubMedGoogle Scholar
  18. Song Y, Noguchi M, Takatsuki K, Sekiguchi T, Mizuno J, Funatsu T, Shoji S, Tsunoda M (2012a) Integration of pillar array columns into a gradient elution system for pressure-driven liquid chromatography. Anal Chem 84(11):4739–4745CrossRefPubMedGoogle Scholar
  19. Song Y, Funatsu T, Tsunoda M (2012b) Rapid determination of amino acids in biological samples using a monolithic silica column. Amino Acids 42(5):1897–1902CrossRefPubMedGoogle Scholar
  20. Song Y, Funatsu T, Tsunoda M (2013a) Amino acid analysis using core-shell particle column. J Chromatogr B 927:214–217CrossRefGoogle Scholar
  21. Song Y, Takatsuki K, Isokawa M, Sekiguchi T, Mizuno J, Funatsu T, Shoji S, Tsunoda M (2013b) Fast and quantitative analysis of branched-chain amino acids in biological samples using a pillar array column. Anal Bioanal Chem 405(25):7993–7999CrossRefPubMedGoogle Scholar
  22. Tsunoda M, Hirayama M, Tsuda T, Ohno K (2015) Noninvasive monitoring of plasma l-dopa concentrations using sweat samples in Parkinson’s disease. Clin Chim Acta 442:52–55CrossRefPubMedGoogle Scholar
  23. Zangerle R, Kurz K, Neurauter G, Kitchen M, Sarcletti M, Fuchs D (2010) Increased blood phenylalanine to tyrosine ratio in HIV-1 infection and correction following effective antiretroviral therapy. Brain Behav Immun 24(3):403–408CrossRefPubMedGoogle Scholar
  24. Zhang L, Majeed B, Lagae L, Peumans P, Van Hoof C, De Malsche W (2013) Ion-pair reversed-phase chromatography of short double-stranded deoxyribonucleic acid in silicon micro-pillar array columns: retention model and applications. J Chromatogr A 1294:1–9CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Wien 2016

Authors and Affiliations

  • Yanting Song
    • 1
    • 2
  • Katsuya Takatsuki
    • 3
  • Tetsushi Sekiguchi
    • 3
  • Takashi Funatsu
    • 1
  • Shuichi Shoji
    • 3
  • Makoto Tsunoda
    • 1
    Email author
  1. 1.Graduate School of Pharmaceutical SciencesUniversity of TokyoTokyoJapan
  2. 2.Key Laboratory of Tropic Biological Resources, Minister of Education, College of Marine ScienceHainan UniversityHaikouChina
  3. 3.Major in Nano-Science and Nano-EngineeringWaseda UniversityTokyoJapan

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