Kuśmierek Krzysztof, Bald Edward
Department of Environmental Chemistry, University of Lodz, Lodz, Poland.
Biomed Chromatogr. 2009 Jul;23(7):770-5. doi: 10.1002/bmc.1183.
A high-performance liquid chromatography method was developed for simultaneous detection and quantitation of total cysteine, glutathione, homocysteine and cysteinylglycine in human plasma. The two key steps in the analysis are reduction of disulfides and treatment with 1-benzyl-2-chloropyridinium bromide, which rapidly and quantitatively reacts with thiol groups to form stable S-pyridinium derivatives with intense UV absorption. The derivatives are well separated on a Zorbax SB C(18) column using reversed-phase high-performance liquid chromatography and monitored at 315 nm. The calibration graphs were linear over concentration ranges covering most experimental and clinical cases with a regression coefficients better than 0.999. The detection and quantitation limits for all analytes were 0.2 and 0.5 micromol/L, respectively. The recoveries were 99.25-101.68%. The intra- and interassay imprecisions were 0.88-4.24 and 1.68-5.14%, respectively. The method was applied for plasma samples donated by apparently healthy volunteers.
建立了一种高效液相色谱法，用于同时检测和定量人血浆中的总半胱氨酸、谷胱甘肽、同型半胱氨酸和半胱氨酰甘氨酸。分析中的两个关键步骤是二硫键的还原以及用1-苄基-2-氯吡啶溴化物处理，该试剂能与巯基快速定量反应，形成具有强烈紫外吸收的稳定S-吡啶鎓衍生物。使用反相高效液相色谱法，这些衍生物在Zorbax SB C(18)柱上得到很好的分离，并在315 nm处进行监测。校准曲线在覆盖大多数实验和临床病例的浓度范围内呈线性，回归系数优于0.999。所有分析物的检测限和定量限分别为0.2和0.5 μmol/L。回收率为99.25 - 101.68%。批内和批间精密度分别为0.88 - 4.24%和1.68 - 5.14%。该方法应用于表面健康志愿者捐献的血浆样本。
