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.
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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.
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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