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Simultaneous Determination of Imidazole Amino Acids, Aromatic Amino Acids, and Creatinine by Dual-Mode Gradient HPLC Using a Low-Capacity Sulfo-Functionalized Poly(Ethylstyrene-Divinylbenzene) Resin Column

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Abstract

This paper presents a low-capacity cation-exchange chromatography method for the analysis of UV-absorbing dipeptides and amino acids. A newly marketed low-capacity cation-exchange column packed with sulfo-functionalized highly cross-linked macroreticular poly(ethylstyrene-divinylbenzene) copolymer was used for the simultaneous determination of imidazole amino acids, aromatic amino acids, and creatinine in urine samples. A dual-mode binary gradient chromatography method was established using two solvents, A: 15 mM H3PO4/5 mM ethylenediamine and B: 15 mM H3PO4/5 mM ethylenediamine/40 (v/v) % CH3CN at 40 °C, with an optimized time program for changing the delivery ratio of A/B and the flow rate. Good chromatograms were obtained within an acceptable cycle time of 25 min. The quantification data were satisfactory for all analytes, showing the relative standard deviations (RSD) of retention times between 0.08 and 1.68 %; RSDs of area intensities between 0.23 and 2.60 %; and linear regression lines with r 2 more than 0.9994. The method could determine the creatinine ratios of the diagnostic markers on the single chromatographic run, which enabled to discriminate disease from health. For example, the creatinine ratios for phenylketonuria were significantly higher than those for controls. The method can provide highly cost-efficient information or useful knowledge for clinical and pharmaceutical studies.

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Acknowledgments

The authors thank Mr. Ryuichi Sugimoto, Mr. Akihiro Shimura, and Miss Yuka Yoshizumi of Mitsubishi Chemical Corporation for helpful discussion in characteristics of the new packing materials and for supplying the analytical columns.

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Authors and Affiliations

  1. Department of Analytical Chemistry, Graduate School of Environment and Information Science, Yokohama National University, 79-7 Tokiwadai, Hodogaya, Yokohama, 240-8501, Japan

    Yukio Yokoyama, Risa Kanazawa & Haruka Ukishima

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  1. Yukio Yokoyama
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  2. Risa Kanazawa
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  3. Haruka Ukishima
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Correspondence to Yukio Yokoyama.

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Yokoyama, Y., Kanazawa, R. & Ukishima, H. Simultaneous Determination of Imidazole Amino Acids, Aromatic Amino Acids, and Creatinine by Dual-Mode Gradient HPLC Using a Low-Capacity Sulfo-Functionalized Poly(Ethylstyrene-Divinylbenzene) Resin Column. Chromatographia 77, 51–57 (2014). https://doi.org/10.1007/s10337-013-2582-8

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  • DOI: https://doi.org/10.1007/s10337-013-2582-8

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