Department of Technological Sciences and Development, University of the Azores, Rua da Mãe de Deus, 9501-801 Ponta Delgada, Azores, Portugal.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Dec 12;911:15-20. doi: 10.1016/j.jchromb.2012.10.022. Epub 2012 Oct 24.
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 (53mm×7mm I.D., 3μm) 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 0.10μmol/L, while LOQ varied from 0.5 to 15μmol/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 6min. By reducing the total run time, a larger number of analysis can be performed daily.
血浆氨基硫醇水平的改变可被视为几种人类疾病（即心血管疾病）诊断和筛查的重要生物标志物。我们旨在优化一种快速、灵敏和准确的反相高效液相色谱法（RP-HPLC），采用荧光检测法，同时测定血浆中半胱氨酸、同型半胱氨酸、半胱氨酰甘氨酸和谷胱甘肽的总浓度，并将其应用于评估一组亚速尔群岛人群的这些巯基水平。采用三正丁基膦将氨基硫醇还原，并用一种巯基特异性荧光衍生试剂铵 7-氟苯并-2-氧代-1,3-二唑-4-磺酸盐对其进行衍生化。采用含 4%乙腈的磷酸盐缓冲液作为流动相，在 Platinum EPS C18 分析柱（53mm×7mm ID，3μm）上进行等度洗脱，分离这些硫醇加合物。结果表明，所有分析物在各自的浓度范围内均具有极好的线性关系，相关系数（r(2)）≥0.99。四种血浆巯基的检测限（LOD）均低于 0.10μmol/L，定量限（LOQ）则在 0.5 至 15μmol/L 之间变化。日内和日间精密度的 RSD（%）值均低于 1.9%，CV（%）值低于 0.5%。回收率在 92%至 100%之间，表明该方法具有很高的准确度。该方法可在 6min 内同时、完全分析四种血浆氨基硫醇和内标物。通过减少总运行时间，可以每天进行更多的分析。
