Determination of ascorbic acid and dehydroascorbic acid in biological samples by high-performance liquid chromatography using subtraction methods: Reliable reduction with tris[2-carboxyethyl] phosphine hydrochloride
Determination of ascorbic acid and dehydroascorbic acid in biological samples by high-performance liquid chromatography using subtraction methods: Reliable reduction with tris[2-carboxyethyl] phosphine hydrochloride
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DOI：
10.1006/abio.2000.4592
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发表时间：
2000-06-15
影响因子：
2.9
通讯作者：
Lykkesfeldt, J
中科院分区：
文献类型：
--
作者：
Lykkesfeldt, J
生物样品中脱氢抗坏血酸的测定最常见的是间接测量，其浓度是用测定的抗坏血酸浓度减去现有脱氢抗坏血酸还原后分析的总抗坏血酸浓度来计算的；一种方法也被称为减法。因此，脱氢抗坏血酸的成功测定依赖于适当的样品处理、化合物的定量还原以及抗坏血酸和总抗坏血酸的准确定量。本文评价了最近引入的三[2-羧乙基]膦(TCEP)作为常用还原剂二硫苏糖醇(DTT)的可靠替代品，结果表明，TCEP在低pH下比DTT更有效地还原脱氢抗坏血酸，而且，尽管DTT保持还原样品环境不到24小时，但TCEP在样品制备后至少96小时完全保护抗坏血酸不被氧化，分析前不需要去除TCEP。提出了一种改进的高效液相色谱-电化学法，以TCEP为还原剂，同时测定了异抗坏血酸和尿酸。对于所有分析物，完整分析的日内和日间变异系数分别小于1.5%和3.5%，总体来说，本文提出的方法比现有方法更简单、更可靠。(C)2000年学术出版社。
Determination of dehydroascorbic acid in biological samples most commonly involves indirect measurement, The concentration is calculated by subtraction of the measured ascorbic acid concentration from that of total ascorbic acid analyzed after reduction of the dehydroascorbic acid present; a methodology also referred to as subtraction methods. Consequently, successful determination of dehydroascorbic acid is dependent on proper sample handling, quantitative reduction of the compound, and accurate quantification of both ascorbic acid and total ascorbic acid. In this paper, the recently introduced reductant tris[2-carboxyethyl]phosphine (TCEP) is evaluated as a reliable alternative to the commonly used reducing agent dithiothreitol (DTT), The results show that TCEP offers a more efficient reduction of dehydroascorbic acid at low pH compared to that of DTT, Moreover, while DTT maintains a reducing sample environment for less than 24 h, TCEP show complete protection from oxidation of ascorbic acid for at least 96 h following sample preparation, Removal of TCEP prior to analysis is unnecessary. A revised HPLC-EC method incorporating TCEP as reductant as well as the co-analysis of isoascorbic acid and uric acid is presented. The within- and between-day coefficients of variation for the complete assay are less than 1.5 and 3.5% for all analytes, As a whole, the method presented here is simpler and more reliable than existing methods. (C) 2000 Academic Press.