Głowacki Rafał, Bald Edward
Department of Environmental Chemistry, University of Lodz, Lodz, Poland.
J Chromatogr B Analyt Technol Biomed Life Sci. 2009 Oct 15;877(28):3400-4. doi: 10.1016/j.jchromb.2009.06.012. Epub 2009 Jun 13.
A fully automated HPLC method for the simultaneous determination of total thiols in plasma samples has been developed. The method involves reductive conversion of disulfides to their reduced counterparts with the use of tris(2-carboxyethyl)phosphine. After reduction the newly formed sulfhydryl groups are reacted with 2-chloro-1-methylquinolinium tetrafluoroborate to form 2-S-quinolinium derivatives followed by deproteinization by dialysis. The reaction products are separated by reversed-phase HPLC, detected and quantified by UV absorbance detection at 355nm. The recommended HPLC procedure enables measurement of four main plasma aminothiols cysteine, cysteinylglycine, glutathione, and homocysteine with low imprecision (mean relative standard deviations within calibration range, 3.47%, 5.34%, 4.25% and 3.26%, respectively) and good sensitivity. Accuracy, expressed as the mean measured amount as percentage of added amount, was within 97.5-103.0%, 98.3-102.5%, 96.3-99.5% and 97.1-99.1%, respectively. The lower limit of quantification for all thiols was 0.5microM. The whole unattended instrument acquisition time amounts 13min.
已开发出一种用于同时测定血浆样品中总硫醇的全自动高效液相色谱（HPLC）方法。该方法包括使用三（2-羧乙基）膦将二硫化物还原转化为其还原形式。还原后，新形成的巯基与四氟硼酸2-氯-1-甲基喹啉鎓反应形成2-S-喹啉鎓衍生物，然后通过透析进行脱蛋白处理。反应产物通过反相HPLC分离，在355nm处通过紫外吸光度检测进行检测和定量。推荐的HPLC程序能够以低不精密度（在校准范围内的平均相对标准偏差分别为3.47%、5.34%、4.25%和3.26%）和良好的灵敏度测量四种主要的血浆氨基硫醇——半胱氨酸、半胱氨酰甘氨酸、谷胱甘肽和同型半胱氨酸。以测得的平均量占添加量的百分比表示的准确度分别在97.5 - 103.0%、98.3 - 102.5%、96.3 - 99.5%和97.1 - 99.1%范围内。所有硫醇的定量下限为0.5微摩尔。整个无人值守的仪器采集时间为13分钟。
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