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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.

12 Issues per year


IMPACT FACTOR 2016: 3.432

CiteScore 2016: 2.21

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1437-4331
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Volume 47, Issue 5 (May 2009)

Issues

Determination of branched chain amino acids, methionine, phenylalanine, tyrosine and α-keto acids in plasma and dried blood samples using HPLC with fluorescence detection

Roman Kand'ár
  • Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Pavla Žáková
  • Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic
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  • De Gruyter OnlineGoogle Scholar
/ Jana Jirošová
  • Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic
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/ Michaela Sladká
  • Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic
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Published Online: 2009-03-16 | DOI: https://doi.org/10.1515/CCLM.2009.123

Abstract

Background: The determination of branched chain amino acids [BCAA; valine (Val), leucine (Leu), isoleucine (Ile)], α-keto acids derived from BCAA [BCKA; α-ketoisovaleric acid (KIV), α-ketoisocaproic acid (KIC), α-ketomethylvaleric acid (KMV)], methionine (Met), phenylalanine (Phe) and tyrosine (Tyr) is currently the most reliable approach for the diagnosis of maple syrup urine disease (MSUD), hypermethioninemia, phenylketonuria (PKU) and tyrosinemia. The aim of this study was to develop rapid and simple HPLC methods for measurement of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples.

Methods: Samples of peripheral venous blood with EDTA as anticoagulant were obtained from a group of healthy blood donors (n=70, 35 females, 27–41 years of age and 35 males, 28–43 years of age). Blood-spot samples from a group of newborns (n=80, 40 girls and 40 boys 3–5 days of age) were collected onto #903 Specimen Collection Paper and allowed to dry for at least 24 h before analysis. Prior to separation, the amino acids (AA) were derivatized with o-phthaldialdehyde (OPA) and BCKA with o-phenylenediamine (OPD). Reverse phase column chromatography (LiChroCart 125-4 Purospher RP-18e, 5 μm) was used for separation and fluorescence detection used to monitoring of effluent. For AA analysis, 25 mmol/L sodium hydrogenphosphate-methanol (90:10, v/v), pH 6.5±0.1 was used as mobile phase A and 100% methanol was used as mobile phase B. Measurement of BCKA used a mixture of methanol and deionized water (55:45, v/v) as mobile phase A and mobile phase B consisted of 100% methanol.

Results: Analytical performance of these methods was satisfactory for the determination of all AA and BCKA. The intra-assay and inter-assay coefficients of variation were below 10% and recovery ranged from 90%–110%.

Conclusions: We have developed simple, rapid and selective HPLC methods with fluorescence detection for the determination of BCAA, Met, Phe, Tyr and BCKA in plasma and dried blood samples.

Clin Chem Lab Med 2009;47:565–72.

Keywords: branched chain amino acids; dried blood spots; HPLC with fluorescence detection; α-keto acids derived from branched chain amino acids

About the article

Corresponding author: Mgr. Roman Kand'ár, PhD, Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Strossova 239, 530 03 Pardubice, Czech Republic Phone: +420 466037715, Fax: +420 466037068,


Received: 2009-01-30

Accepted: 2009-02-03

Published Online: 2009-03-16

Published in Print: 2009-05-01


Citation Information: Clinical Chemistry and Laboratory Medicine, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/CCLM.2009.123.

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©2009 by Walter de Gruyter Berlin New York. Copyright Clearance Center

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