Determination of N-Acetylcysteine and Main Endogenous Thiols in Human Plasma by HPLC with Ultraviolet Detection in the Form of Their S-Quinolinium Derivatives
Determination of N-Acetylcysteine and Main Endogenous Thiols in Human Plasma by HPLC with Ultraviolet Detection in the Form of Their S-Quinolinium Derivatives
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DOI：
10.1080/10826070903249666
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发表时间：
2009-01-01
影响因子：
1.3
通讯作者：
Bald, E.
中科院分区：
文献类型：
--
作者：
Glowacki, R.;Bald, E.
建立了人血浆中总N-乙酰半胱氨酸、谷胱甘氨酸、谷胱甘肽和同型半胱氨酸的高效液相色谱检测方法。用2-氯-1-甲基喹啉四氟硼酸盐柱前特异衍生法测定硫醇浓度，然后用高效液相色谱反相分离-分光光度法检测。使用还原剂三(2-羧乙基)膦将硫醇的氧化和蛋白质结合形式转化为其还原形式。分离时间为12.5min，批内、批间精密度均小于10%。洗脱曲线如下：0-4分钟，11%B；4-8分钟，11-30%B；8-12分钟，30-11%B。使用添加了越来越多的分析物的正常血浆获得的校正图在浓度范围内是线性的，覆盖了大多数实验和临床病例(同型半胱氨酸和谷胱甘肽为1-32亩M，N-乙酰半胱氨酸和半胱氨酸为1-320亩M，半胱氨酸和半胱氨酸为1-亩M)。对于所有分析物，回收率在91.2%至108.6%之间。
A new, sensitive, repeatable, and robust high performance liquid chromatography assay for the determination of total N-acetylcysteine, cysteine, cysteinylglycine, glutathione, and homocysteine in human plasma has been developed. The thiol concentrations were measured using precolumn specific derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate, followed by high performance liquid chromatographic reversed phase separation with spectrophotometric detection. The oxidized and protein bound forms of thiols were converted into their reduced forms employing a reductive agent tris(2-carboxyethyl) phosphine. The chromatographic separation was accomplished in 12.5 min; the within run and between run imprecision were all less than 10%. The elution profile was as follows: 0-4 min, 11% B; 4-8 min, 11-30% B; 8-12 min, 30-11% B. The calibration graphs, obtained with the use of normal plasma spiked with growing amounts of analytes, were linear over the concentration ranges, covering most experimental and clinical cases (1-32 mu M for homocysteine and glutathione, 1-320 mu M for N-acetylcysteine and cysteine, and 1-64 mu M for cysteinylglycine). For all analytes, recoveries between 91.2 and 108.6%, were observed.