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Amino Acids

, Volume 47, Issue 9, pp 2005–2014 | Cite as

Analysis of l-homoarginine in biological samples by HPLC involving precolumn derivatization with o-phthalaldehyde and N-acetyl-l-cysteine

  • Yongqing Hou
  • Sichao Jia
  • Gayan Nawaratna
  • Shengdi Hu
  • Sudath Dahanayaka
  • Fuller W. Bazer
  • Guoyao WuEmail author
Original Article
Part of the following topical collections:
  1. Homoarginine, Arginine and Relatives

Abstract

l-Homoarginine (hArg) may play a role in regulating the metabolism of its structural homologue l-arginine via multiple pathways (including nitric oxide synthase) in animals. Accurate measurement of hArg is essential for studying its synthesis and utilization by cells and the whole body. Here, we describe a simple, sensitive and automated method for analysis of hArg in biological samples by high-performance liquid chromatography involving precolumn derivatization with o-phthalaldehyde (OPA) and N-acetyl-l-cysteine (NAC) as the thiol. The hArg–OPA–NAC derivative was separated at 25 °C on a reversed-phase C18 material and detected by fluorescence at excitation and emission wavelengths of 340 and 450 nm, respectively. The total running time for one sample (including the time for column regeneration) was 20 min, with the retention time for hArg being 10.03 min. The limit of detection was 188 fmol hArg, which was equivalent to 12 nM hArg in the 160-µl assay mixture. The assay was linear between 1.0 and 80 pmol hArg injected into the HPLC column (equivalent to 0.0625 and 5 µM hArg in the 160-µl assay mixture, respectively). The precision (relative deviation, %) and bias (relative error, %) of the HPLC method were 0.52–1.16 and 0.42–1.12, respectively, for aqueous solutions of hArg and for various biological samples (e.g., plasma, liver, brain and kidney). This is a highly sensitive, accurate, efficient and easily automated method for analysis of hArg in biological samples and provides a useful tool for studying the biochemistry, nutrition, physiology and pharmacology of hArg and arginine in animals and humans.

Keywords

l-Homoarginine Derivatization o-Phthalaldehyde N-Acetyl-l-cysteine HPLC 

Abbreviations

AA

Amino acids

AGAT

l-Arginine:glycine amidinotransferase

hArg

l-Homoarginine

HPLC

High-performance liquid chromatography

LC

Liquid chromatography

LOD

Limit of detection

MS

Mass spectrometry

NAC

N-Acetyl-l-cysteine

NOS

Nitric oxide synthase

OPA

o-Phthaldialdehyde

SPE

Solid-phase extraction

Notes

Acknowledgments

This research was supported by National Basic Research Program of China (No. 2012CB126305), National Natural Science Foundation of China (No. 31372319, 31402084), the Hubei Provincial Key Project for Scientific and Technical Innovation (2014ABA022), Hubei Provincial Research and Development Program (No. 2010BB023), Natural Science Foundation of Hubei Province (No. 2013CFA097, 2013CFB325, 2012FFB04805, 2011CDA131), Scientific Research Program of Hubei Provincial Department of Education (D20141701), the Hubei Hundred Talent Program, Agriculture and Food Research Initiative Competitive Grants (2011-67015-20028 and 2014-67015-21770) of the USDA National Institute of Food and Agriculture and Texas A&M AgriLife Research (H-8200).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics statement

The use of animals for this research was approved by the Institutional Animal Care and Use Committee of Texas A&M University.

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Copyright information

© Springer-Verlag Wien 2015

Authors and Affiliations

  • Yongqing Hou
    • 1
    • 2
  • Sichao Jia
    • 2
  • Gayan Nawaratna
    • 2
  • Shengdi Hu
    • 2
  • Sudath Dahanayaka
    • 2
  • Fuller W. Bazer
    • 2
  • Guoyao Wu
    • 1
    • 2
    Email author
  1. 1.Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed SafetyWuhan Polytechnic UniversityWuhanChina
  2. 2.Department of Animal ScienceTexas A&M UniversityCollege StationUSA

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