A validated HPLC-fluorescence method with a semi-micro column for routine determination of homocysteine, cysteine and cysteamine, and the relation between the thiol derivatives in normal human plasma.
A validated HPLC-fluorescence method with a semi-micro column for routine determination of homocysteine, cysteine and cysteamine, and the relation between the thiol derivatives in normal human plasma.
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DOI：
10.1002/bmc.1205
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发表时间：
2009-09
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通讯作者：
S. Ichinose;Mayumi Nakamura;Mayu Maeda;Rie Ikeda;M. Wada;M. Nakazato;Y. Ohba;N. Takamura;T. Maeda;K. Aoyagi;K. Nakashima
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作者：
S. Ichinose;Mayumi Nakamura;Mayu Maeda;Rie Ikeda;M. Wada;M. Nakazato;Y. Ohba;N. Takamura;T. Maeda;K. Aoyagi;K. Nakashima
描述了一种用于常规测定硫醇衍生物如同型半胱氨酸 (Hcy)、半胱氨酸 (Cys) 和半胱胺 (CA) 的半微柱 HPLC 荧光方法。用 7-氟苯并-2-氧杂-1,3-二唑-4-磺酸铵 (SBD-F) 标记的硫醇衍生物在半微量 ODS 柱 (Daisopak-SP-120-5-ODS-BP) 上以 25 mm 乙酸盐缓冲液 (pH 2.00) 和 CH(3)CN 的混合物作为流动相，在 12 分钟内进行等度分离。多波长荧光检测仪检测SBD-硫醇的纯度和相似度分别达到92.3%和96.7%以上。信噪比为3时Hcy、Cys和CA的检测限分别为0.16、0.47和0.03μm。此外，所提出方法的准确性、精密度和鲁棒性等验证参数显示出令人满意的结果。可以对一根色谱柱进行近 850 次血浆样品注射（范围 572-1076，n = 3），而标签的保留时间和峰高没有差异。作为该方法的应用，演示了正常人血浆（n = 103）中硫醇衍生物的测定。 Hcy与Cys和Hcy与CA之间的相关系数分别为0.38和-0.35。
A semi-micro column HPLC-fluorescence method for routine determination of thiol derivatives such as homocysteine (Hcy), cysteine (Cys) and cysteamine (CA) is described. The thiol derivatives labeled with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) were isocratically separated within 12 min on a semi-micro ODS column (Daisopak-SP-120-5-ODS-BP) with a mixture of 25 mm acetate buffer (pH 2.00) and CH(3)CN as a mobile phase. The purity and similarity of SBD-thiols by a multi-wavelength fluorescence detector were more than 92.3 and 96.7%. The detection limits of Hcy, Cys and CA at a signal-to-noise ratio of 3 were 0.16, 0.47 and 0.03 microm, respectively. Furthermore validation parameters such as accuracy, precision and robustness of the proposed method showed satisfactory results. Almost 850 plasma sample injections (range 572-1076, n = 3) for a column could be performed without differences in retention time and peak heights of labels. As an application of the proposed method, the determination of thiol derivatives in normal human plasma (n = 103) was demonstrated. The correlation coefficients between Hcy vs Cys and Hcy vs CA were 0.38 and -0.35, respectively.