Department of Chemistry and Industrial Chemistry, University of Genoa, Via Dodecaneso, 31-16146 Genoa, Italy.
J Chromatogr A. 2010 Apr 2;1217(14):2158-62. doi: 10.1016/j.chroma.2010.02.012. Epub 2010 Feb 10.
Altered levels of aminothiols in biological fluids are thought to be an important risk indicator for several diseases, and reliable methods for the accurate determination of aminothiols concentrations in plasma are thus required. In this paper ammonium 5-bromo-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-BF) is proposed as a convenient fluorogenic derivatizating reagent for the determination of aminothiols (cysteine, cysteinylglycine, homocysteine and glutathione) by HPLC with fluorescence detection. The reactions of SBD-BF with aminothiols at room temperature are about three-times faster than those of ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (the most frequently employed reagent) at 60 degrees C. The derivatives of SBD-BF with cysteine, cysteinylglycine, homocysteine and glutathione are easily separated by HPLC and their calibration curves show excellent linearity over the range 0.05-20 micromol/L with excellent r(2) values for all analytes. SBD-BF reacts with thiols under mild conditions, i.e. at 25 degrees C over about 30 min, and is proposed as a suitable fluorogenic reagent for thiol derivatization to be introduced in analytical clinical chemistry. The detection limits of Cys, Cys-Gly, Hcy and GSH at a signal-to-noise ratio of 5 were 0.1 microM for Cys, 0.01 microM for Cys-Gly and Hcy, and 0.02 microM for GSH. Furthermore, validation parameters of the proposed method are quite satisfactory. As an application of this method the determination of thiol derivatives in human plasma was carried out on a number of samples.
生物体液中氨基硫醇水平的改变被认为是几种疾病的重要风险指标，因此需要可靠的方法来准确测定血浆中氨基硫醇的浓度。在本文中，我们提出了一种方便的荧光衍生化试剂，即铵 5-溴-7-氟苯并-2-氧代-1,3-二唑-4-磺酸盐（SBD-BF），用于通过 HPLC 与荧光检测法测定氨基硫醇（半胱氨酸、半胱氨酰甘氨酸、同型半胱氨酸和谷胱甘肽）。SBD-BF 与氨基硫醇在室温下的反应速度比 60°C 时铵 7-氟苯并-2-氧代-1,3-二唑-4-磺酸盐（最常使用的试剂）快约 3 倍。SBD-BF 与半胱氨酸、半胱氨酰甘氨酸、同型半胱氨酸和谷胱甘肽的衍生物很容易通过 HPLC 分离，它们的校准曲线在 0.05-20µmol/L 范围内具有极好的线性，所有分析物的 r(2)值均很好。SBD-BF 在温和条件下（即在 25°C 下约 30 分钟）与硫醇反应，被提议作为一种合适的荧光衍生试剂，用于引入分析临床化学中的硫醇衍生化。在信噪比为 5 时，Cys、Cys-Gly、Hcy 和 GSH 的检测限分别为 0.1µM、0.01µM 和 0.02µM。此外，该方法的验证参数非常令人满意。作为该方法的应用，我们对一些人血浆样品中的硫醇衍生物进行了测定。
