Methodology for a rapid and simultaneous determination of total cysteine, homocysteine, cysteinylglycine and glutathione in plasma by isocratic RP-HPLC
Methodology for a rapid and simultaneous determination of total cysteine, homocysteine, cysteinylglycine and glutathione in plasma by isocratic RP-HPLC
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DOI：
10.1016/j.jchromb.2012.10.022
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发表时间：
2012-12-12
影响因子：
3
通讯作者：
Baptista, Jose
中科院分区：
文献类型：
--
作者：
Ferin, Rita;Pavao, Maria Leonor;Baptista, Jose
血浆氨基硫醇水平的变化可被认为是诊断和筛查几种人类疾病的重要生物标志物，即心血管疾病。我们旨在优化一种快速、灵敏、准确的荧光检测反相高效液相色谱方法，同时测定血浆中半胱氨酸、同型半胱氨酸、半胱氨酸甘氨酸和谷胱甘肽的总浓度，并将其应用于一组氮卓类受试者血浆中这些硫醇水平的评估。用三正丁基膦还原氨基硫醇，用硫醇特异的荧光试剂7-氟苯并-2-氧杂-1，3-二唑-4-磺酸铵进行衍生化。硫醇加合物以含4%乙腈的磷酸盐缓冲液为流动相，在铂金EPS C18分析柱(53 mm×7 mm i.d.，3微米)上进行等度洗脱分离。结果表明，所有分析物在各自的浓度范围内都具有良好的线性关系，相关系数r(2)>；=0.99。四种硫醇的检出限均小于0.10mU/L，检出限为0.5~15mU/L，日内和日间精密度RSD(%)均小于1.9%，CV(%)均小于0.5%。回收率在92%~100%之间，具有较高的准确度。这种方法可以在6分钟内同时完整地分析四种血浆中的氨基硫醇和内标。通过减少总运行时间，每天可以执行更多的分析。(C)2012爱思唯尔B.V.保留所有权利。
Alterations in the plasma aminothiols levels can be considered as important biomarkers for the diagnosis and screening of several human disorders, namely cardiovascular diseases. We aimed to optimize a rapid, sensitive and accurate RP-HPLC methodology with fluorescence detection, for the simultaneous determination of the total concentrations of cysteine, homocysteine, cysteinylglycine and glutathione in blood plasma, as well as its application to the evaluation of those thiols levels in plasma of a group of Azorean subjects. Aminothiols were reduced with tri-n-butylphosphine and derivatized with a thiol-specific fluorogenic reagent ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate. The thiols adducts were separated by an isocratic elution on a Platinum EPS C18 analytical column (53 mm x 7 mm I.D., 3 mu m) using a phosphate buffer containing 4% of acetonitrile as a mobile phase. Results indicated an excellent linearity for all the analytes over their respective concentration ranges with correlation coefficients (r(2)) >= 0.99. The LOD for the four plasma thiols was lower than 0.10 mu mol/L, while LOQ varied from 0.5 to 15 mu mol/L. For both intra- and inter-day precision, the RSD (%) values were lower than 1.9%, and the CV (%) values were found under 0.5%. The recovery ranged from 92% to 100% indicating a high degree of the method's accuracy. This method allows a simultaneous, complete analysis of the four plasma aminothiols and the internal standard in 6 min. By reducing the total run time, a larger number of analysis can be performed daily. (C) 2012 Elsevier B.V. All rights reserved.